Fluorescent Nucleobase Conjugates Having Anionic Linkers

ABSTRACT

Provided are nucleotide-dye conjugates and related compounds in which a dye is linked to a nucleobase directly or indirectly by an anionic linker. The anionic character of the linker is provided by one or more anionic moieties which are present in the linker, such as phosphate, phosphonate, sulfonate, and carboxylate groups. When the dye is a provided as a donor/acceptor dye pair, the anionic linker can be located between the donor and the acceptor, or between the nucleobase and either the donor or acceptor, or both. In one embodiment, conjugates of the invention provide enhanced electrophoretic mobility characteristics to sequencing fragments, e.g., for dideoxy sequencing using labeled terminators.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/977,341, filed Oct. 28, 2004, which is a continuation of U.S. patentapplication Ser. No. 09/976,168, filed Oct. 11, 2001, now, U.S. Pat. No.6,811,979, which claims the benefit of U.S. Provisional PatentApplication No. 60/239,660, filed Oct. 11, 2000, which are allincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to fluorescent dye compounds, and toconjugates and uses thereof. The invention also relates to fluorescentpolynucleotide conjugates having improved electrophoretic mobilities.

INTRODUCTION

The analysis of complex mixtures of polynucleotides is important in manybiological applications. In many situations, it is necessary to separatecomponents of such mixtures to detect target polynucleotides ofinterest, to determine relative amounts of different components, and toobtain nucleotide sequence information, for example.

Electrophoresis provides convenient methods for analyzingpolynucleotides. Typically, polynucleotides can be separated on thebasis of length, due to differences in electrophoretic mobility. Forexample, in a matrix such as crosslinked polyacrylamide, polynucleotidestypically migrate at rates that are inversely proportional topolynucleotide length, due to size-dependent obstruction by thecrosslinked matrix. In free solution, polynucleotides tend to migrate atsubstantially the same rates because of their substantially identicalmass to charge ratios, so that it is difficult to distinguish differentpolynucleotides based on size alone. However, distinguishableelectrophoretic mobilities can be obtained in free solution usingpolynucleotides that contain different charge/mass ratios, e.g., byattaching to the polynucleotides a polymer or other chemical entityhaving a charge/mass ratio that differs from that of the polynucleotidesalone (e.g., see U.S. Pat. No. 5,470,705).

When different polynucleotides can be separated based on length ormolecular weight, detection can usually be accomplished using a singledetectable label, such as a radioisotope or fluorophore. However, incomplex mixtures or when different-sequence polynucleotides have similaror identical mobilities, it is preferable to use two or more detectablelabels to distinguish different polynucleotides unambiguously.

In DNA sequencing, it is now conventional to use two or more (usuallyfour) different fluorescent labels to distinguish sequencing fragmentsthat terminate with one of the four standard nucleotide bases (A, C, Gand T, or analogs thereof). Such labels are usually introduced into thesequencing fragments using suitably labeled extension primers(dye-primer method) or by performing primer extension in the presence ofnonextendable nucleotides that contain unique labels (Sanger dideoxyterminator method). Electrophoresis of the labeled products generatesladders of fragments that can be detected on the basis of elution timeor band position.

Under sieving conditions in crosslinked or non-crosslinked matrices,shorter polynucleotide fragments migrate more rapidly than longerfragments. Usually, the inter-band spacing and migration rates offragments decrease gradually in proportion to increasing length.However, anomalous migration patterns can occur due tosequence-dependent secondary structures within fragments, even in thepresence of denaturing agents such as urea. For example, poly-G segmentsoften cause band compression that make sequence determination of theseregions difficult. Compressed band regions can often be resolved usingnucleotide analogs such as dITP (2′-deoxyinosine-5′-triphosphate) or7-deaza-dGTP in the extension reaction instead of dGTP, or by sequencingthe complementary strand.

Anomalous migration patterns may also occur for polynucleotide fragmentsthat contain a detectable label, due to interactions between the labeland one or more bases in the polynucleotide. Such interactions can beparticularly problematic when the interactions are sequence-dependent,so that different-sequence fragments having the same-lengths may havesignificantly different mobilities. This phenomenon can be inconvenientfor sequencing, especially in automated sequencing methods. Accordingly,there is a need for labeled compounds and methods of use to improveelectrophoretic performance.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a conjugate comprising adye-labeled nucleobase of the form: (1) B-L-D, wherein B is anucleobase, L is an anionic linker, and D comprises at least onefluorescent dye, or (2) B-L1-D1-L2-D2, wherein B is a nucleobase, L1 andL2 are linkers such that at least one of L1 and L2 is an anionic linker,and D1 and D2 are members of a fluorescent donor/acceptor pair, suchthat one of D1 and D2 is a donor dye capable of absorbing light at afirst wavelength and emitting excitation energy in response thereto, andthe other of D1 and D2 is an acceptor dye capable of absorbing theexcitation energy emitted by the donor dye and fluorescing at a secondwavelength in response thereto.

Each anionic linker may contain one or more anionic groups, such as asulfonic acid moiety, a sulfate monoester, an anionic phosphate, ananionic phosphonate, or a carboxylic acid. In one embodiment, L, L1 orL2 contains a phosphate diester moiety whose phosphorus atom is locatedwithin a chain of linker atoms (bridging position) or can be asubstituent attached to a chain of linker atoms (non-bridging position).In another embodiment, the linker contains a monoanionic phosphonateester which can be located within the linker chain or attached to thelinker chain. Other embodiments are described further herein.

In embodiments in which the conjugate has the form B-L1-D1-L2-D2, one ofL1 and L2 can be a nonanionic linker. In one embodiment, L1 is ananionic linker and L2 is non-anionic. For example, when L2 isnon-anionic, D1-L2-D2 may comprise structure (a), (b) or (c) below:

-   -   (a) -D1-R₂₁Z₁(O)R₂₂R₂₈-D2    -   (b) -D1-R₂₈R₂₂C(O)Z₁R₂₁-D2    -   (c) -D1-R₂₈R₂₂R₂₈-D2        wherein: R₂₁ is C₁-C₅ alkyldiyl, Z₁ is NH, S, or O, R₂₂ is        ethenediyl, ethynediyl, 1,3-butadienediyl, 1,3-butadiynediyl, a        5- or 6-membered ring having at least one unsaturated bond or a        fused ring structure having at least one unsaturated bone, and        R₂₈ is a bond or spacer group (a linking segment) that links R22        to D1 or D2. In another embodiment, L1 can be a nonionic linker,        of which the following are examples: —C≡CCH₂NH—,        —C≡CCH₂NHC(O)(CH₂)₅NH—, —C═CC(O)NH(CH₂)₅NH—, —C≡CCH₂OCH₂CH₂NH—,        —C≡CCH₂OCH₂CH₂OCH₂CH₂NH—, —C≡C—CH₂OCH₂CH₂—NH—, and        —C≡C(p-C₄H₆)OCH₂CH₂NH—.

Fluorescent dyes used in accordance with the invention can include anyfluorescent compound suitable for the purposes of the present invention.Typically, each dye comprises a conjugated, resonance-delocalized oraromatic ring system that absorbs light at a first wavelength and emitslight at a second wavelength in response thereto. For example, the dyescan be selected independently from any of a variety of classes offluorescent compounds, such as xanthene, rhodamine, dibenzorhodamine,fluorescein, [8,9]benzophenoxazine, cyanine, phthalocyanine, squaraine,or bodipy dye.

In another aspect, the invention includes a labeled nucleosidetriphosphate comprising a conjugate of the type described herein. In oneembodiment, the labeled nucleoside triphosphate is not 3′-extendable.For example, the labeled nucleoside triphosphate can be a2′,3′-dideoxynucleotide or 3′-fluoro-2′,3′-dideoxynucleotide. In anotherembodiment, the labeled nucleoside triphosphate is extendable andcontains a 3′-hydroxyl group.

In another aspect, the invention includes a polynucleotide comprising aconjugate of the type discussed herein. In one embodiment, the conjugateis located in a 3′ terminal nucleotide subunit of a polynucleotide, suchthat the subunit may be extendable or nonextendable. In anotherembodiment, the conjugate is located on a non-terminal nucleotidesubunit.

In a further embodiment, the invention provides a mixture comprising aplurality of different-sequence polynucleotides, wherein at least onepolynucleotide contains a conjugate as described herein. In oneembodiment, the mixture comprises at least two different-sequencepolynucleotides which each contain a different conjugate to identify theattached polynucleotide. In another embodiment, the mixture comprisesfour classes of polynucleotides, wherein the polynucleotides in eachclass terminate with a different terminator subunit type that contains adistinct nucleobase-dye conjugate to identify the polynucleotides inthat class.

The invention also includes a method of identifying one or morepolynucleotide(s). In the method, one or more labeled different-sequencepolynucleotides are formed such that each different-sequencepolynucleotide contains a unique conjugate as described herein. The oneor more labeled different-sequence polynucleotides are separated byelectrophoresis on the basis of size, and one or more different-sequencepolynucleotides are identified on the basis of electrophoreticmobilities and fluorescence properties.

The invention also provides a method of forming a labeled polynucleotidestrand, the method comprising reacting together (i) a duplexpolynucleotide comprising a 3′-extendable strand hybridized to acomplementary template strand having a 5′ overhang, (ii) atemplate-dependent polymerase enzyme, and (iii) a labeled nucleosidetriphosphate containing a conjugate as described herein, underconditions effective to form a labeled polynucleotide containing theconjugate. In one embodiment, the labeled nucleoside triphosphate isnonextendable. In another embodiment, the labeled nucleosidetriphosphate is extendable.

The invention also provides a method of sequencing a targetpolynucleotide sequence. In the method, four classes of polynucleotidesare formed which are complementary to a target polynucleotide sequence,by template-dependent primer extension, wherein the polynucleotides ineach class terminate with a different terminator subunit type thatcontains a distinct nucleobase-dye conjugate to identify thepolynucleotides in that class. The resultant polynucleotides areseparated on the basis of size to obtain a mobility pattern from whichthe sequence of the target polynucleotide sequence can be determined.

The invention also provides kits for performing the various methods ofthe invention. For nucleic acid sequencing, the kit comprises at leastone labeled nucleoside triphosphate comprising a conjugate describedherein. The kit may also include one or more of the followingcomponents: a 3′-extendable primer, a polymerase enzyme, one or more 3′extendable nucleotides which are not labeled with conjugate, and/or abuffering agent. In some embodiments, the kit includes at least onelabeled nucleoside triphosphate that is nonextendable. In otherembodiments, the kit comprises four different labeled nucleosidetriphosphates which are complementary to A, C, T and G, and each ofwhich contains a distinct conjugate as described herein. In yet anotherembodiment, the labeled nucleoside triphosphates are nonextendable. Inanother embodiment, the labeled nucleoside triphosphates are extendableribonucleoside triphosphates. In another embodiment, the kit comprisesat least one labeled, nonextendable nucleoside triphosphate comprising aconjugate described herein, and one or more of the following components:a 3′-extendable primer, a polymerase enzyme, and/or a buffering agent.

These and other objects and features of the invention will become moreapparent from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-18B and 22A-22B illustrate exemplary synthetic protocols forpreparing various conjugates in accordance with the invention.

FIGS. 19A and 19B show electropherograms of sequencing laddersterminating with a first set of terminators (ddG).

FIGS. 20A and 20B show electropherograms of sequencing laddersterminating with a second set of terminators different from those inFIGS. 1A and 1B (ddA).

FIGS. 21A and 21B show electropherograms of sequencing laddersterminating with a third set of terminators (ddA).

DETAILED DESCRIPTION

The present invention is directed to novel dye compounds and dyeconjugates that have various advantageous properties. The invention hasgeneral application in the areas of fluorescent nucleic acid analysis,e.g., automated DNA or RNA sequencing, fragment analysis, detection ofnucleic acid amplification products, detection of probe hybridization inhybridization arrays, diagnostic tests, and the like. In one aspect, theinvention provides polynucleotides having more consistent size-dependentelectrophoretic mobilities, such that sequence-dependent anomalies arereduced or eliminated. The invention finds application in automatedsequencing methods which rely on uniform, size-dependent electrophoreticmobilities to determine whether low peak signals should be included ordiscarded, and whether overlapping peaks represent fragments of the samelength. The invention is also useful in sequencing methods that involvethe formation of 3′ dye-labeled sequencing fragments. In addition, theinvention can be used in polynucleotide detection and identificationmethods that rely on absolute or relative migration times or migrationdistances for polynucleotide identification.

I. DEFINITIONS

Unless stated otherwise, the following terms and phrases used herein areintended to have the following meanings:

“Alkyl” refers to a saturated or unsaturated, branched, straight-chainor cyclic monovalent hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane, alkene oralkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“alkenyl” and/or “alkynyl” is used, as defined below. In preferredembodiments, the alkyl groups are (C₁-C₆) alkyl.

“Alkanyl” refers to a saturated branched, straight-chain or cyclic alkylradical derived by the removal of one hydrogen atom from a single carbonatom of a parent alkane. Typical alkanyl groups include, but are notlimited to, methanyl; ethanyl; propanyls such as propan-1-yl,propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butyanyls such asbutan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like. Inpreferred embodiments, the alkanyl groups are (C₁-C₆) alkanyl.

“Alkenyl” refers to an unsaturated branched, straight-chain or cyclicalkyl radical having at least one carbon-carbon double bond derived bythe removal of one hydrogen atom from a single carbon atom of a parentalkene. The radical may be in either the cis or trans conformation aboutthe double bond(s). Typical alkenyl groups include, but are not limitedto, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, prop-2-en-2-yl, cycloprop-1-en-1-yl;cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl,2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like. Inpreferred embodiments, the alkenyl group is (C₂-C₆) alkenyl.

“Alkynyl” refers to an unsaturated branched, straight-chain or cyclicalkyl radical having at least one carbon-carbon triple bond derived bythe removal of one hydrogen atom from a single carbon atom of a parentalkyne. Typical alkynyl groups include, but are not limited to, ethynyl;propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such asbut-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Inpreferred embodiments, the alkynyl group is (C₂-C₆) alkynyl.

“Alkyldiyl” refers to a saturated or unsaturated, branched,straight-chain or cyclic divalent hydrocarbon radical derived by theremoval of one hydrogen atom from each of two different carbon atoms ofa parent alkane, alkene or alkyne, or by the removal of two hydrogenatoms from a single carbon atom of a parent alkane, alkene or alkyne.The two monovalent radical centers or each valency of the divalentradical center can form bonds with the same or different atoms. Typicalalkyldiyls include, but are not limited to methandiyl; ethyldiyls suchas ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Inpreferred embodiments, the alkyldiyl group is (C₁-C₆) alkyldiyl. Alsopreferred are saturated acyclic alkanyldiyl radicals in which theradical centers are at the terminal carbons, e.g., methandiyl (methano);ethan-1,2-diyl (ethano); propan-1,3-diyl (propano); butan-1,4-diyl(butano); and the like (also referred to as alkylenos, defined infra).

“Alkyleno” refers to a straight-chain alkyldiyl radical having twoterminal monovalent radical centers derived by the removal of onehydrogen atom from each of the two terminal carbon atoms ofstraight-chain parent alkane, alkene or alkyne. Typical alkyleno groupsinclude, but are not limited to, methano; ethylenos such as ethano,etheno, ethyno; propylenos such as propano, prop[1]eno, propa[1,2]dieno,prop[1]yno, etc.; butylenos such as butano, but[1]eno, but[2]eno,buta[1,3]dieno, but[1]yno, but[2]yno, but[1,3]diyno, etc.; and the like.Where specific levels of saturation are intended, the nomenclaturealkano, alkeno and/or alkyno is used. In preferred embodiments, thealkyleno group is (C₁-C₆) alkyleno.

“Heteroalkyl, Heteroalkanyl, Heteroalkenyl, Heteroalkanyl,Heteroalkyldiyl and Heteroalkyleno” refer to alkyl, alkanyl, alkenyl,alkynyl, alkyldiyl and alkyleno radicals, respectively, in which one ormore of the carbon atoms are each independently replaced with the sameor different heteroatomic groups. Typical heteroatomic groups which canbe included in these radicals include, but are not limited to, —O—, —S—,—O-—O—, —S—S—, —O—S—, —NR′—, ═N—N═, —N═N—, —N(O)N—, —N═N—NR′—, —PH—,—P(O)₂—, —O—P(O)₂—, —SH₂—, —S(O)₂—, —SnH₂— and the like, where each R′is independently hydrogen, alkyl, alkanyl, alkenyl, alkynyl, aryl,arylaryl, arylalkyl, heteroaryl, heteroarylalkyl orheteroaryl-heteroaryl as defined herein.

“Acyclic Heteroatomic Bridge” refers to a divalent bridge in which thebackbone atoms are exclusively heteroatoms. Typical acyclic heteroatomicbridges include, but are not limited to, any of the various heteroatomicgroups listed above, either alone or in combinations.

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated □ electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, indane, indene, phenalene, etc. Typical parent aromaticring systems include, but are not limited to, aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene,hexylene, as-indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene,and the like.

“Aryl” refers to a monovalent aromatic hydrocarbon radical derived bythe removal of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, radicals derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. In preferredembodiments, the aryl group is (C₅-C₁₄) aryl, with (C₅-C₁₀) being evenmore preferred. Particularly preferred aryls are phenyl and naphthyl.

“Aryldiyl” refers to a divalent aromatic hydrocarbon radical derived bythe removal of one hydrogen atom from each of two different carbon atomsof a parent aromatic ring system or by the removal of two hydrogen atomsfrom a single carbon atom of a parent aromatic ring system. The twomonovalent radical centers or each valency of the divalent center canform bonds with the same or different atom(s). Typical aryldiyl groupsinclude, but are not limited to, divalent radicals derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexylene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene, and the like. In preferred embodiments, the aryldiylgroup is (C₅-C₁₄) aryldiyl, with (C₅-C₁₀) being even more preferred. Themost preferred aryldiyl groups are divalent radicals derived frombenzene and naphthalene, especially phena-1,4-diyl, naphtha-2,6-diyl andnaphtha-2,7-diyl.

“Aryleno” refers to a divalent bridge radical having two adjacentmonovalent radical centers derived by the removal of one hydrogen atomfrom each of two adjacent carbon atoms of a parent aromatic ring system.Attaching an aryleno bridge radical, e.g. benzeno, to a parent aromaticring system, e.g. benzene, results in a fused aromatic ring system, e.g.naphthalene. The bridge is assumed to have the maximum number ofnon-cumulative double bonds consistent with its attachment to theresultant fused ring system. In order to avoid double-counting carbonatoms, when an aryleno substituent is formed by taking together twoadjacent substituents on a structure that includes alternativesubstituents, the carbon atoms of the aryleno bridge replace thebridging carbon atoms of the structure. As an example, consider thefollowing structure

Wherein R¹, when taken alone is hydrogen, or when taken together with R²is (C₅-C₁₄) aryleno; and R², when taken alone is hydrogen, or when takentogether with R¹ is (C₅-C₁₄) aryleno.

When R¹ and R² are each hydrogen, the resultant compound is benzene.When R¹ taken together with R² is C₆ aryleno (benzeno), the resultantcompound is naphthalene. When R¹ taken together with R² is C₁₀ aryleno(naphthaleno), the resultant compound is anthracene or phenanthrene.Typical aryleno groups include, but are not limited to, aceanthryleno,acenaphthyleno, acephenanthryleno, anthraceno, azuleno, benzeno (benzo),chryseno, coroneno, fluorantheno, fluoreno, hexaceno, hexapheno,hexyleno, as-indaceno, s-indaceno, indeno, naphthaleno (naphtho),octaceno, octapheno, octaleno, ovaleno, penta-2,4-dieno, pentaceno,pentaleno, pentapheno, peryleno, phenaleno, phenanthreno, piceno,pleiadeno, pyreno, pyranthreno, rubiceno, triphenyleno, trinaphthaleno,and the like. Where a specific connectivity is intended, the involvedbridging carbon atoms (of the aryleno bridge) are denoted in brackets,e.g., [1,2]benzeno ([1,2]benzo), [1,2]naphthaleno, [2,3]naphthaleno,etc. Thus, in the above example, when R¹ taken together with R² is[2,3]naphthaleno, the resultant compound is anthracene. When R¹ takentogether with R² is [1,2]naphthaleno, the resultant compound isphenanthrene. In a preferred embodiment, the aryleno group is (C₅-C₁₄),with (C₅-C₁₀) being even more preferred.

“Arylaryl” refers to a monovalent hydrocarbon radical derived by theremoval of one hydrogen atom from a single carbon atom of a ring systemin which two or more identical or non-identical parent aromatic ringsystems are joined directly together by a single bond, where the numberof such direct ring junctions is one less than the number of parentaromatic ring systems involved. Typical arylaryl groups include, but arenot limited to, biphenyl, triphenyl, phenyl-naphthyl, binaphthyl,biphenyl-naphthyl, and the like. When the number of carbon atomscomprising an arylaryl group is specified, the numbers refer to thecarbon atoms comprising each parent aromatic ring. For example, (C₅-C₁₄)arylaryl is an arylaryl group in which each aromatic ring comprises from5 to 14 carbons, e.g., biphenyl, triphenyl, binaphthyl, phenylnaphthyl,etc. Preferably, each parent aromatic ring system of an arylaryl groupis independently a (C₅-C₁₄) aromatic, more preferably a (C₅-C₁₀)aromatic. Also preferred are arylaryl groups in which all of the parentaromatic ring systems are identical, e.g., biphenyl, triphenyl,binaphthyl, trinaphthyl, etc.

“Biaryl” refers to an arylaryl radical having two identical parentaromatic systems joined directly together by a single bond. Typicalbiaryl groups include, but are not limited to, biphenyl, binaphthyl,bianthracyl, and the like. Preferably, the aromatic ring systems are(C₅-C₁₄) aromatic rings, more preferably (C₅-C₁₀) aromatic rings. Aparticularly preferred biaryl group is biphenyl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Where specific allyl moieties are intended, the nomenclaturearylalkanyl, arylakenyl and/or arylalkynyl is used. In preferredembodiments, the arylalkyl group is (C₆-C₂₀) arylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₆) andthe aryl moiety is (C₅-C₁₄). In particularly preferred embodiments thearylalkyl group is (C₆-C₁₃), e.g., the alkanyl, alkenyl or alkynylmoiety of the arylalkyl group is (C₁-C₃) and the aryl moiety is(C₅-C₁₀).

“Parent Heteroaromatic Ring System” refers to a parent aromatic ringsystem in which one or more carbon atoms (and any necessary associatedhydrogen atoms) are each independently replaced with the same ordifferent heteroatom. Typical heteroatoms to replace the carbon atomsinclude, but are not limited to, N, P, O, S, Si, etc. Specificallyincluded within the definition of “parent heteroaromatic ring systems”are fused ring systems in which one or more rings are aromatic and oneor more of the rings are saturated or unsaturated, such as, for example,arsindole, chromane, chromene, indole, indoline, xanthene, etc. Typicalparent heteroaromatic ring systems include, but are not limited to,arsindole, carbazole, -carboline, chromane, chromene, cinnoline, furan,imidazole, indazole, indole, indoline, indolizine, isobenzofuran,isochromene, isoindole, isoindoline, isoquinoline, isothiazole,isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike.

“Heteroaryl” refers to a monovalent heteroaromatic radical derived bythe removal of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, radicals derived from acridine, arsindole, carbazole,-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. In preferred embodiments,the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 memberedheteroaryl being particularly preferred. The most preferred heteroarylradicals are those derived from parent heteroaromatic ring systems inwhich any ring heteroatoms are nitrogens, such as imidazole, indole,indazole, isoindole, naphthyridine, pteridine, isoquinoline,phthalazine, purine, pyrazole, pyrazine, pyridazine, pyridine, pyrrole,quinazoline, quinoline, etc.

“Heteroaryldiyl” refers to a divalent heteroaromatic radical derived bythe removal of one hydrogen atom from each of two different atoms of aparent heteroaromatic ring system or by the removal of two hydrogenatoms from a single atom of a parent heteroaromatic ring system. The twomonovalent radical centers or each valency of the single divalent centercan form bonds with the same or different atom(s). Typicalheteroaryldiyl groups include, but are not limited to, divalent radicalsderived from acridine, arsindole, carbazole, -carboline, chromane,chromene, cinnoline, furan, imidazole, indazole, indole, indoline,indolizine, isobenzofuran, isochromene, isoindole, isoindoline,isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole,oxazole, perimidine, phenanthridine, phenanthroline, phenazine,phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine,pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline,quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene,triazole, xanthene, and the like. In preferred embodiments, theheteroaryldiyl group is 5-14 membered heteroaryldiyl, with 5-10 memberedbeing particularly preferred. The most preferred heteroaryldiyl groupsare divalent radicals derived from parent heteroaromatic ring systems inwhich any ring heteroatoms are nitrogens, such as imidazole, indole,indazole, isoindole, naphthyridine, pteridine, isoquinoline,phthalazine, purine, pyrazole, pyrazine, pyridazine, pyridine, pyrrole,quinazoline, quinoline, etc.

“Heteroaryleno” refers to a divalent bridge radical having two adjacentmonovalent radical centers derived by the removal of one hydrogen atomfrom each of two adjacent atoms of a parent heteroaromatic ring system.Attaching a heteroaryleno bridge radical, e.g. pyridino, to a parentaromatic ring system, e.g. benzene, results in a fused heteroaromaticring system, e.g., quinoline. The bridge is assumed to have the maximumnumber of non-cumulative double bonds consistent with its attachment tothe resultant fused ring system. In order to avoid double-counting ringatoms, when a heteroaryleno substituent is formed by taking together twoadjacent substituents on a structure that includes alternativesubstituents, the ring atoms of the heteroaryleno bridge replace thebridging ring atoms of the structure. As an example, consider thefollowing structure

wherein R¹, when taken alone is hydrogen, or when taken together with R²is 5-14 membered heteroaryleno; and R², when taken alone is hydrogen, orwhen taken together with R¹ is 5-14 membered heteroaryleno.

When R¹ and R² are each hydrogen, the resultant compound is benzene.When R¹ taken together with R² is a 6-membered heteroaryleno (e.g.,pyridino), the resultant compound is isoquinoline, quinoline orquinolizine. When R¹ taken together with R² is a 10-memberedheteroaryleno (e.g., isoquinoline), the resultant compound is, e.g.,acridine or phenanthridine. Typical heteroaryleno groups include, butare not limited to, acridino, carbazolo, -carbolino, chromeno,cinnolino, furano, imidazolo, indazoleno, indoleno, indolizino,isobenzofurano, isochromeno, isoindoleno, isoquinolino, isothiazoleno,isoxazoleno, naphthyridino, oxadiazoleno, oxazoleno, perimidino,phenanthridino, phenanthrolino, phenazino, phthalazino, pteridino,purino, pyrano, pyrazino, pyrazoleno, pyridazino, pyridino, pyrimidino,pyrroleno, pyrrolizino, quinazolino, quinolino, quinolizino,quinoxalino, tetrazoleno, thiadiazoleno, thiazoleno, thiopheno,triazoleno, xantheno, and the like. Where a specific connectivity isintended, the involved bridging atoms (of the heteroaryleno bridge) aredenoted in brackets, e.g., [1,2]pyridino, [2,3]pyridino, [3,4]pyridino,etc. Thus, in the above example, when R¹ taken together with R² is[1,2]pyridino, the resultant compound is quinolizine. When R¹ takentogether with R² is [2,3]pyridino, the resultant compound is quinoline.When R¹ taken together with R² is [3,4]pyridino, the resultant compoundis isoquinoline. In preferred embodiments, the heteroaryleno group is5-14 membered heteroaryleno, with 5-10 membered being even morepreferred. The most preferred heteroaryleno radicals are those derivedfrom parent heteroaromatic ring systems in which any ring heteroatomsare nitrogens, such as imidazolo, indolo, indazolo, isoindolo,naphthyridino, pteridino, isoquinolino, phthalazino, purino, pyrazolo,pyrazino, pyridazino, pyridino, pyrrolo, quinazolino, quinolino, etc.

“Heteroaryl-Heteroaryl” refers to a monovalent heteroaromatic radicalderived by the removal of one hydrogen atom from a single atom of a ringsystem in which two or more identical or non-identical parentheteroaromatic ring systems are joined directly together by a singlebond, where the number of such direct ring junctions is one less thanthe number of parent heteroaromatic ring systems involved. Typicalheteroaryl-heteroaryl groups include, but are not limited to, bipyridyl,tripyridyl, pyridylpurinyl, bipurinyl, etc. When the number of ringatoms are specified, the numbers refer to the number of atoms comprisingeach parent heteroatomatic ring systems. For example, 5-14 memberedheteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which eachparent heteroaromatic ring system comprises from 5 to 14 atoms, e.g.,bipyridyl, tripyridyl, etc. Preferably, each parent heteroaromatic ringsystem is independently a 5-14 membered heteroaromatic, more preferablya 5-10 membered heteroaromatic. Also preferred are heteroaryl-heteroarylgroups in which all of the parent heteroaromatic ring systems areidentical. The most preferred heteroaryl-heteroaryl radicals are thosein which each heteroaryl group is derived from parent heteroaromaticring systems in which any ring heteroatoms are nitrogens, such asimidazole, indole, indazole, isoindole, naphthyridine, pteridine,isoquinoline, phthalazine, purine, pyrazole, pyrazine, pyridazine,pyridine, pyrrole, quinazoline, quinoline, etc.

“Biheteroaryl” refers to a heteroaryl-heteroaryl radical having twoidentical parent heteroaromatic ring systems joined directly together bya single bond. Typical biheteroaryl groups include, but are not limitedto, bipyridyl, bipurinyl, biquinolinyl, and the like. Preferably, theheteroaromatic ring systems are 5-14 membered heteroaromatic rings, morepreferably 5-10 membered heteroaromatic rings. The most preferredbiheteroaryl radicals are those in which the heteroaryl groups arederived from a parent heteroaromatic ring system in which any ringheteroatoms are nitrogens, such as biimidazolyl, biindolyl, biindazolyl,biisoindolyl, binaphthyridinyl, bipteridinyl, biisoquinolinyl,biphthalazinyl, bipurinyl, bipyrazolyl, bipyrazinyl, bipyridazinyl,bipyridinyl, bipyrrolyl, biquinazolinyl, biquinolinyl, etc.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Where specific alkylmoieties are intended, the nomenclature heteroarylalkanyl,heteroarylakenyl and/or heterorylalkynyl is used. In preferredembodiments, the heteroarylalkyl group is a 6-20 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of theheteroarylalkyl is 1-6 membered and the heteroaryl moiety is a5-14-membered heteroaryl. In particularly preferred embodiments, theheteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl,alkenyl or alkynyl moiety is 1-3 membered and the heteroaryl moiety is a5-10 membered heteroaryl.

“Substituted” refers to a radical in which one or more hydrogen atomsare each independently replaced with the same or differentsubstituent(s). Typical substituents include, but are not limited to,—X, —R, —O⁻, ═O, —OR, —SR, —S⁻, ═S, —NRR, ═NR, perhalo (C₁-C₆) alkyl,—CX₃, —CF₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R, —C(S)X, —C(O)OR, —C(O)O⁻,—C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and —C(NR)NRR, where eachX is independently a halogen (preferably —F or —Cl) and each R isindependently hydrogen, alkyl, alkanyl, alkenyl, alkynyl, aryl,arylalkyl, arylaryl, heteroaryl, heteroarylalkyl orheteroaryl-heteroaryl, carboxyl, acetyl, sulfonyl, sulfinyl, sulfone,phosphate, or phosphonate, as defined herein. The actual substituentsubstituting any particular group will depend upon the identity of thegroup being substituted.

“Nucleobase” means a nitrogen-containing heterocyclic moiety capable offorming Watson-Crick type hydrogen bonds with a complementary nucleobaseor nucleobase analog, e.g. a purine, a 7-deazapurine, or a pyrimidine.Typical nucleobases are the naturally occurring nucleobases adenine,guanine, cytosine, uracil, thymine, and analogs of naturally occurringnucleobases, e.g. 7-deazaadenine, 7-deaza-8-azaadenine, 7-deazaguanine,7-deaza-8-azaguanine, inosine, nebularine, nitropyrrole, nitroindole,2-amino-purine, 2,6-diamino-purine, hypoxanthine, pseudouridine,pseudocytidine, pseudoisocytidine, 5-propynyl-cytidine, isocytidine,isoguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine,4-thiouracil, O⁶-methylguanine, N⁶-methyl-adenine, O⁴-methylthymine,5,6-dihydrothymine, 5,6-dihydrouracil, 4-methylindole, and ethenoadenine(Fasman, Practical Handbook of Biochemistry and Molecular Biology, pp.385-394, CRC Press, Boca Raton, Fl (1989)).

“Nucleoside” means a compound comprising a nucleobase linked to a C-1′carbon of a ribose sugar or analog thereof. The ribose or analog may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those riboses in which one or more of the carbon atoms,preferably the 3′-carbon atom, is substituted with one or more of thesame or different substituents such as —R, —OR, —NRR or halogen (e.g.,fluoro, chloro, bromo, or iodo), where each R group is independently —H,C₁-C₆ alkyl or C₃-C₁₄ aryl. Particularly preferred riboses are ribose,2′-deoxyribose, 2′,3′-dideoxyribose, 3′-haloribose (such as3′-fluororibose or 3′-chlororibose) and 3′-alkylribose. Typically, whenthe nucleobase is A or G, the ribose sugar is attached to theN⁹-position of the nucleobase. When the nucleobase is C, T or U, thepentose sugar is attached to the N¹-position of the nucleobase (Kornbergand Baker, DNA Replication, 2^(nd) Ed., Freeman, San Francisco, Calif.,(1992)). Examples of ribose analogs include arabinose, 2′-O-methylribose, and locked nucleoside analogs (e.g., WO 99/14226), for example,although many other analogs are also known in the art.

“Nucleotide” means a phosphate ester of a nucleoside, either as anindependent monomer or as a subunit within a polynucleotide. Nucleotidetriphosphates are sometimes denoted as “NTP”, “dNTP” (2′-deoxypentose)or “ddNTP” (2′,3′-dideoxypentose) to particularly point out thestructural features of the ribose sugar. “Nucleotide 5′-triphosphate”refers to a nucleotide with a triphosphate ester group at the 5′position. The triphosphate ester group may include sulfur substitutionsfor one or more phosphate oxygen atoms, e.g. α-thionucleotide5′-triphosphates.

“Polynucleotide” and “oligonucleotide”, which are used interchangeablyherein, refer to linear polymers of natural nucleotide monomers oranalogs thereof, including for example, double- and single-strandeddeoxyribonucleotides, ribonucleotides, □-anomeric forms thereof, and thelike. A polynucleotide may be composed entirely of deoxyribonucleotides,ribonucleotides, or analogs thereof, or may contain blocks or mixturesof two or more different monomer types. Usually nucleoside monomers arelinked by phosphodiester linkages. However, polynucleotides andoligonucleotides containing non-phosphodiester linkages are alsocontemplated. “Polynucleotide” and “oligonucleotide” also encompasspolymers that contain one or more non-naturally occurring monomersand/or intersubunit linkages, such as peptide nucleic acids (PNAs, e.g.,polymers comprising a backbone of amide-linked N-(2-aminoethyl)-glycinesubunits to which nucleobases are attached via the non-amide backbonenitrogens. See Nielsen et al., Science 254:1497-1500 (1991)).Polynucleotides typically range in size from a few monomeric units, e.g.8-40, to several thousand monomeric units. Whenever a polynucleotide isrepresented by a sequence of letters, such as “ATGCCTG,” it will beunderstood that the nucleotides are in 5′->3′ order from left to rightand that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G”denotes deoxyguanosine, and “T” denotes thymidine, unless otherwisenoted.

“Nucleotide subunit” or “polynucleotide subunit” refers to a singlenucleotide or nucleotide analog within a polynucleotide orpolynucleotide analog.

“Phosphate analog” refers to an analog of phosphate wherein one or moreof the oxygen atoms is replaced with a non-oxygen moiety. Exemplaryphosphate analogs including phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphotriester, phosphoranilidate, phosphoramidate, alkylphosphonatessuch as methylphosphonates, boronophosphates.

“Linker” refers to a moiety that links a dye to a substrate such as anoligonucleotide, or links one dye to another dye (e.g., links a donor toan acceptor dye).

“Enzymatically incorporatable” means that a nucleotide is capable ofbeing enzymatically incorporated onto the terminus, e.g. 3′ terminus, ofa polynucleotide chain, or internally through nick-translation of apolynucleotide chain, through action of a template-dependent ortemplate-independent polymerase enzyme. A nucleotide-5′-triphosphate isan example of an enzymatically incorporatable nucleotide.

“Enzymatically extendable” or “3′extendable” means a nucleotide orpolynucleotide that is capable of being appended to a nucleotide orpolynucleotide by enzyme action. A polynucleotide containing a 3′hydroxyl group is an example of an enzymatically extendablepolynucleotide.

“Terminator” means an enzymatically incorporatable nucleotide whichprevents subsequent incorporation of nucleotides to the resultingpolynucleotide chain and thereby halts polymerase-mediated extension.Typical terminators lack a 3′-hydroxyl substituent and include2′,3′-dideoxyribose, 2′,3′-didehydroribose, and2′,3′-dideoxy-3′-haloribose, e.g. 3′-deoxy-3′-fluoro-ribose or2′,3′-dideoxy-3′-fluororibose, for example. Alternatively, aribofuranose analog can be used, such as2′,3′-dideoxy-□-D-ribofuranosyl, □-D-arabinofuranosyl,3′-deoxy-□-D-arabinofuranosyl, 3′-amino-2′,3′-dideoxy-□-D-ribofuranosyl,and 2′,3′-dideoxy-3′-fluoro-□-D-ribofuranosyl (see, for example,Chidgeavadze et al., Nucleic Acids Res., 12: 1671-1686 (1984), andChidgeavadze et al. FEB. Lett., 183: 275-278 (1985)). Nucleotideterminators also include reversible nucleotide terminators (Metzker etal. Nucleic Acids Res., 22(20):4259 (1994)).

“Nonextendable” or “3′nonextendable” refers to the fact that aterminator is incapable, or substantially incapable, of being extendedin the 3′ direction by a template-dependent DNA or RNA polymerase.

“Spectrally resolvable” means that two or more dyes have emission bandsthat are sufficiently distinct, i.e., sufficiently non-overlapping, thatthey can be distinguished on the basis of a unique fluorescent signalgenerated by each dye.

Generally, whenever a compound mentioned in this disclosure contains apositive or negative charge, it should be understood that such compoundmay also be accompanied by a suitable counterion that balances thepositive or negative charge. Exemplary positively charged counterionsinclude, without limitation, H⁺, NH₄ ⁺, Na⁺, K⁺, Mg²⁺, trialkylammonium(such as triethylammonium), tetraalkylammonium (such astetraethylammonium), and the like. Exemplary negatively chargedcounterions include, without limitation, carbonate, bicarbonate,acetate, chloride, and phosphate, for example. Also, although particularresonance structures may be shown herein, such structures are intendedto include all other possible resonance structures.

II. CONJUGATES

In one aspect, the present invention provides compositions that compriseat least one dye-labeled nucleobase of the type described herein. Suchcompositions include not only nucleobase-dye conjugates as independentmolecules, but also as nucleosides, nucleotides and polynucleotidescontaining such conjugates.

In one embodiment, a dye-labeled nucleobase of the invention has theform B-L-D, wherein B is a nucleobase, L is an anionic linker, and D isa fluorescent dye.

Nucleobase B may be any moiety capable of forming Watson-Crick hydrogenbonds with a complementary nucleobase or nucleobase analog, as set forthin the Definition section above. Typically, B is a nitrogen-containingheterocyclic moiety such as a 7-deazapurine, purine, or pyrimidinenucleotide base. In certain embodiments, B is uracil, cytosine,7-deazaadenine, or 7-deazaguanosine. When B is a purine, the linker isusually attached to the 8-position of the purine. When B is a7-deazapurine, the linker to the dye is usually attached to the7-position of the 7-deazapurine. When B is pyrimidine, the linker isusually attached to the 5-position of the pyrimidine.

Fluorescent dye D may be any fluorescent dye which is suitable for thepurposes of the invention. Typically, the fluorescent dye comprises aresonance-delocalized system or aromatic ring system that absorbs lightat a first wavelength and emits fluorescent light at a second wavelengthin response to the absorption event. A wide variety of such dyemolecules are known in the art. For example, fluorescent dyes can beselected from any of a variety of classes of fluorescent compounds, suchas xanthenes, rhodamines, fluoresceins, cyanines, phthalocyanines,squaraines, and bodipy dyes.

In one embodiment, the dye is a xanthene-type dye, which contains afused three-ring system of the form:

This parent xanthene ring may be unsubstituted (i.e., all substituentsare H) or may be substituted with one or more of a variety of the sameor different substituents, such as described below.

In one embodiment, the dye contains a parent xanthene ring having thegeneral structure:

In the parent xanthene ring depicted above, A¹ is OH or NH₂ and A² is Oor NH₂ ⁺. When A¹ is OH and A² is O, the parent xanthene ring is afluorescein-type xanthene ring. When A¹ is NH₂ and A² is NH₂ ⁺, theparent xanthene ring is a rhodamine-type xanthene ring. When A¹ is NH₂and A² is O, the parent xanthene ring is a rhodol-type xanthene ring. Inthe parent xanthene ring depicted above, one or both nitrogens of A¹ andA² (when present) and/or one or more of the carbon atoms at positionsC1, C2, C4, C5, C7, C8 and C9 can be independently substituted with awide variety of the same or different substituents. In one embodiment,typical substituents include, but are not limited to, —X, —R, —OR, —SR,—NRR, perhalo (C₁-C₆) alkyl, —CX₃, —CF₃, —CN, —OCN, —SCN, —NCO, —NCS,—NO, —NO₂, —N₃, —S(O)₂O⁻, —S(O)₂OH, —S(O)₂R, —C(O)R, —C(O)X, —C(S)R,—C(S)X, —C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRRand —C(NR)NRR, where each X is independently a halogen (preferably —F orCl) and each R is independently hydrogen, (C₁-C₆) allyl, (C₁-C₆)alkanyl, (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, (C₅-C₂₀) aryl, (C₆-C₂₆)arylalkyl, (C₅-C₂₀) arylaryl, heteroaryl, 6-26 membered heteroarylalkyl5-20 membered heteroaryl-heteroaryl, carboxyl, acetyl, sulfonyl,sulfinyl, sulfone, phosphate, or phosphonate. Moreover, the C1 and C2substituents and/or the C7 and C8 substituents can be taken together toform substituted or unsubstituted buta[1,3]dieno or (C₅-C₂₀) arylenobridges. Generally, substituents which do not tend to quench thefluorescence of the parent xanthene ring are preferred, but in someembodiments quenching substituents may be desirable. Substituents thattend to quench fluorescence of parent xanthene rings areelectron-withdrawing groups, such as —NO₂, —Br, and —I. In oneembodiment, C9 is unsubstituted. In another embodiment, C9 issubstituted with a phenyl group. In another embodiment, C9 issubstituted with a substituent other than phenyl.

When A¹ is NH₂ and/or A² is NH₂ ⁺, these nitrogens can be included inone or more bridges involving the same nitrogen atom or adjacent carbonatoms, e.g., (C₁-C₁₂) alkyldiyl, (C₁-C₁₂) alkyleno, 2-12 memberedheteroalkyldiyl and/or 2-12 membered heteroalkyleno bridges.

Any of the substituents on carbons C1, C2, C4, C5, C7, C8, C9 and/ornitrogen atoms at C3 and/or C6 (when present) can be further substitutedwith one or more of the same or different substituents, which aretypically selected from —X, —R′, ═O, —OR′, —SR′, ═S, —NR′R′, ═NR′, —CX₃,—CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂, —N₃, —NHOH, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R′, —P(O)(O⁻)₂, —P(O)(OH)₂, —C(O)R′, —C(O)X, —C(S)R′,—C(S)X, —C(O)OR′, —C(O)O⁻, —C(S)OR′, —C(O)SR′, —C(S)SR′, —C(O)NR′R′,—C(S)NR′R′ and —C(NR)NR′R′, where each X is independently a halogen(preferably —F or —Cl) and each R′ is independently hydrogen, (C₁-C₆)allyl, 2-6 membered heteroalkyl, (C₅-C₁₄) aryl or heteroaryl, carboxyl,acetyl, sulfonyl, sulfinyl, sulfone, phosphate, or phosphonate.

Exemplary parent xanthene rings include, but are not limited to,rhodamine-type parent xanthene rings and fluorescein-type parentxanthene rings.

In one embodiment, the dye contains a rhodamine-type xanthene dye thatincludes the following ring system:

In the rhodamine-type xanthene ring depicted above, one or bothnitrogens and/or one or more of the carbons at positions C1, C2, C4, C5,C7 or C8 can be independently substituted with a wide variety of thesame or different substituents, as described above for the parentxanthene rings, for example. Exemplary rhodamine-type xanthene dyesinclude, but are not limited to, the xanthene rings of the rhodaminedyes described in U.S. Pat. Nos. 5,936,087, 5,750,409, 5,366,860,5,231,191, 5,840,999, 5,847,162, and 6,080,852 (Lee et al.), PCTPublications WO 97/36960 and WO 99/27020, Sauer et al., J. Fluorescence5(3):247-261 (1995), Arden-Jacob, Neue Lanwellige Xanthen-Farbstoffe fürFluoreszenzsonden und Farbstoff Laser, Verlag Shaker, Germany (1993),and Lee et al., Nucl. Acids Res. 20:2471-2483 (1992). Also includedwithin the definition of “rhodamine-type xanthene ring” are theextended-conjugation xanthene rings of the extended rhodamine dyesdescribed in U.S. application Ser. No. 09/325,243 filed Jun. 3, 1999.

In another embodiment, the dye comprises a fluorescein-type parentxanthene ring having the structure:

In the fluorescein-type parent xanthene ring depicted above, one or moreof the carbons at positions C1, C2, C4, C5, C7, C8 and C9 can beindependently substituted with a wide variety of the same or differentsubstituents, as described above for the parent xanthene rings.Exemplary fluorescein-type parent xanthene rings include, but are notlimited to, the xanthene rings of the fluorescein dyes described in U.S.Pat. Nos. 4,439,356, 4,481,136, 5,188,934, 5,654,442, and 5,840,999, WO99/16832, and EP 050684. Also included within the definition of“fluorescein-type parent xanthene ring” are the extended xanthene ringsof the fluorescein dyes described in U.S. Pat. Nos. 5,750,409 and5,066,580.

In another embodiment, the dye comprises a rhodamine dye, whichcomprises a rhodamine-type xanthene ring in which the C9 carbon atom issubstituted with an orthocarboxy phenyl substituent (pendent phenylgroup). Such compounds are also referred to herein asorthocarboxyfluoresceins. A particularly preferred subset of rhodaminedyes are 4,7,-dichlororhodamines. Typical rhodamine dyes include, butare not limited to, rhodamine B, 5-carboxyrhodamine, rhodamine X (ROX),4,7-dichlororhodamine X (dROX), rhodamine 6G (R6G),4,7-dichlororhodamine 6G, rhodamine 110 (R110), 4,7-dichlororhodamine110 (dR110), tetramethyl rhodamine (TAMRA) and4,7-dichloro-tetramethylrhodamine (dTAMRA). Additional rhodamine dyescan be found, for example, in U.S. Pat. Nos. 5,366,860 (Bergot et al.),5,847,162 (Lee et al.), 6,017,712 (Lee et al.), 6,025,505 (Lee et al.),6,080,852 (Lee et al.), 5,936,087 (Benson et al.), 6,111,116 (Benson etal.), 6,051,719 (Benson et al.), 5,750,409, 5,366,860, 5,231,191,5,840,999, and 5,847,162, U.S. application Ser. No. 09/325,243 filedJun. 3, 1999, PCT Publications WO 97/36960 and WO 99/27020, Sauer etal., 1995, J. Fluorescence 5(3):247-261, Arden-Jacob, 1993, NeueLanwellige Xanthen-Farbstoffe für Fluoresenzsonden und Farbstoff Laser,Verlag Shaker, Germany, and Lee et al., Nucl. Acids Res.20(10):2471-2483 (1992), Lee et al., Nucl. Acids Res. 25:2816-2822(1997), and Rosenblum et al., Nucl. Acids Res. 25:4500-4504 (1997), forexample. In one embodiment, the dye is a4,7-dichloro-orthocarboxyrhodamine.

In another embodiment, the dye comprises a fluorescein dye, whichcomprises a fluorescein-type xanthene ring in which the C9 carbon atomis substituted with an orthocarboxy phenyl substituent (pendent phenylgroup). A preferred subset of fluorescein-type dyes are4,7,-dichlorofluoresceins. Typical fluorescein dyes include, but are notlimited to, 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM).Additional typical fluorescein dyes can be found, for example, in U.S.Pat. Nos. 5,750,409, 5,066,580, 4,439,356, 4,481,136, 5,188,934 (Menchenet al.), 5,654,442 (Menchen et al.), 6,008,379 (Benson et al.), and5,840,999, PCT publication WO 99/16832, and EPO Publication 050684. Inone embodiment, the dye is a 4,7-dichloro-orthocarboxyfluorescein.

In other embodiments, the dye can be a cyanine, phthalocyanine,squaraine, or bodipy dye, such as described in the following referencesand references cited therein: U.S. Pat. No. 5,863,727 (Lee et al.), U.S.Pat. No. 5,800,996 (Lee et al.), U.S. Pat. No. 5,945,526 (Lee et al.),U.S. Pat. No. 6,080,868 (Lee et al.), U.S. Pat. No. 5,436,134 (Hauglandet al.), U.S. Pat. No. 5,863,753 (Haugland et al.), U.S. Pat. No.6,005,113 (Wu et al.), and WO 96/04405 (Glazer et al.).

Sometimes, the designation -1 or -2 is placed after an abbreviation of aparticular dye, e.g., HEX-1. The “-1” and “-2” designations indicatethat a particular 5- or 6-carboxy dye isomer being used. The 1 and 2isomers are defined by order of elution (the 1 isomer elutes before the2 isomer) of free dye in a reverse-phase chromatographic separationsystem utilizing a C-8 column and an elution gradient of 15%acetonitrile/85% 0.1 M triethylammonium acetate to 35% acetonitrile/65%0.1 M triethylammonium acetate.

Anionic linker L is designed to have an overall negative charge.Typically, this negative charge is provided by one or more negativelycharged groups in the linker. If the anionic linker contains apositively charged group (e.g., bridging or nonbridging amino groups),then the linker must also contain a sufficient number of negativelycharged groups to ensure that the total negative charge in the linkerexceeds the total positive charge. In one embodiment, the linkercontains no positively charged groups. The linker may contain one, two,or more net negative charges which may be provided by one, two, or morenegatively charged groups. In one embodiment, the linker contains asingle negative charge. In another specific embodiment, the linkercontains two negative charges which may be provided by a single group ortwo groups. In specific embodiments, the overall charge of the linker atpH 9 can be at least −1, −2, −3, or greater. Preferably, the overallcharge of the linker at pH 9 is at least −1. By way of non-limitingexamples, such anionic groups include: phosphate monoester (—OPO₃ ²⁻),phosphodiester of the form —OP(═O)(O⁻)O— (in which the phosphorus andtwo oxygen atoms are linker chain atoms), phosphodiester of the form—OP(═O)(O⁻)(OR) (in which R is a masking group such as alkyl, alkenyl,alkynyl, aryl, alkaryl, etc., and the left-most oxygen is bound directlyor indirectly to the linker chain), phosphonate monoester of the form—Y—P(═O)(O⁻)O— (in which Y is an alkyl carbon, such as CH₂, an ethenecarbon, an ethyne carbon, or a benzene ring carbon, and preferably CH₂),and Y, the phosphorus atom and the right-hand oxygen atom are linkerchain atoms), phosphonate monoester of the form —Y—P(═O)(O⁻)(OR) or—OP(═O)(O⁻)(Z) (wherein Y is as just described, and R and Z are eachmasking groups as just defined for R, but none of the phosphonatemonoester atoms are linker chain atoms), sulfate monoester (—OSO₃ ⁻),sulfonic acid (sulfonate, —SO₃ ⁻), and carboxylic acid (carboxylate,—CO₂ ⁻). In addition, anionic groups can include groups with apK_(a)<10, such as nitrophenolate, thiolate, phenylthiolate, fluorinatedalkyl alcohol (e.g., perfluoro hydroxymethyl or perfluoro hydroxyethyl),sulfonimides, and squarates.

Anionic groups within a linker can be classified in various ways. First,anionic groups can be divided between bridging and non-bridging groups,depending on whether an anionic group is within the chain of linkeratoms (a bridging anionic group) or is outside the chain of linker atoms(a non-bridging group). Examples of bridging anionic groups arephosphodiester of the form —OP(═O)(O⁻)O—, and phosphonate monoester ofthe form —CH₂P(═O)(O⁻)O—. Examples of non-bridging anionic groups arephosphate monoester (—OPO₃ ²⁻), phosphodiester of the form —OP(═O)(O⁻)OR(where R is defined as above), phosphonic acid (—PO₃ ²⁻), phosphonatemonoester of the form —CH₂P(═O)(O⁻)(OR) or —OP(═O)(O⁻)(Z) (where R and Zare defined as above), sulfate monoester, sulfonic acid, and carboxylicacid. Accordingly, the invention contemplates linkers that contain oneor more bridging anionic groups, one or more divalent anionic groups,and combinations thereof.

Anionic groups can also be classified by net formal charge. Examples ofgroups that provide a single negative charge at pH 9 includephosphodiester (both bridging and non-bridging), phosphonate monoesterof the form —CH₂P(═O)(O⁻)O—, —CH₂P(═O)(O⁻)(OR), and —OP(═O)(O⁻)(Z),sulfate monoester, sulfonic acid, and carboxylic acid. Examples ofgroups that provide a double negative charge at pH 9 include phosphatemonoester (non-bridging) and phosphonic acid of the form (—CH₂PO₃ ²⁻).Accordingly, the invention contemplates linkers that contain singlycharged anionic groups, doubly charged anionic groups, and combinationsthereof.

Any of a variety of anionic linkers can be used. Typically, a linkerbetween B and D will have a linker chain length of from about 4 to about30 linker chain atoms, and typically from 4 to 20 linker chain atoms,although shorter and longer linkers may also be used. Several exemplaryanionic linkers are illustrated in the attached Figures and in thecompounds prepared in the Examples below.

The junction between a nucleobase B and linker L can be located at anysuitable position on the nucleobase. Preferably, the attachment site onthe nucleobase is selected so as not to interfere with or eliminate theH-bonding capability of the nucleobase with respect to a complementarynucleobase. When B includes a purine nucleobase, the linker is usuallyattached to the N-8-position of the purine. When B includes a7-deazapurine nucleobase, the linker is usually attached to theN-7-position of the 7-deazapurine. When B includes a pyrimidine base,the linkage is attached to the C-5-position of the pyrimidine. In anucleoside, nucleotide, or polynucleotide subunit, a purine or7-deazapurine is usually attached to a sugar moiety via the N-9-positionof the purine or deazapurine, and a pyrimidine is usually attached to asugar moiety via the N-1-position of the pyrimidine.

The particular entity by which a linker is connected to a nucleobase canbe any chemical group that is suitable for the purposes of the presentinvention. A variety of suitable chemical groups are known. For example,the terminal chemical group in the linker that is covalently attached tothe nucleobase can be an acetylene moiety (—C≡C—), and often is apropargyl moiety (—C≡CCH₂—), since such linkage moieties tend to beparticularly compatible with a variety of polymerase enzymes used forprimer extension. However, non-acetylenic chemical groups are alsocontemplated. Examples of suitable terminal groups for attachment to anucleobase can be found in the following exemplary references:

TABLE 1 Aminopropargyl EPO Patent No. 251786B1 (Hobbs et al.) U.S. Pat.Nos. 5,151,507 and 5,047,519 (Hobbs et al.) Hobbs et al., J. Org. Chem.,54: 3420 (1989). Oxypropargyl or U.S. Pat. No. 5,821,356 (Khan et al.)aminoethyloxypropargyl U.S. Pat. No. 5,770,716 (Khan et al.) U.S. Pat.No. 5,936,087 (Benson et al.) —(C≡C)_(n)—Ar_(o)—C≡C_(p)— U.S. Pat. No.5,948,648 (Khan et al.) and variants thereof U.S. Pat. No. 6,096,875(Khan et al.) Acylethenyl U.S. Pat. No. 6,080,852 (Lee et al.)—C≡C—C₆H₄— U.S. Pat. No. 6,080,852 (Lee et al.) Other U.S. Pat. No.6,008,379 (Benson et al.)

The junction between linker L and dye moiety D can be located at anysuitable position on the dye moiety, preferably so that the fluorescentproperties of the dye are not adversely affected. For a xanthene-typering, the linker can be joined to any available carbon atom, or to oneof the nitrogen atoms in a rhodamine-type xanthene ring. For a rhodaminedye or fluorescein dye, the substituent positions on the pendent phenylring are also available, particularly the positions which are para to C9of the xanthene ring (5 position), or para to the ortho carboxyl group(6 position). In addition, the particular chemical group by which alinker is connected to a nucleobase can be any chemical group that issuitable for the purposes of the present invention. A variety ofchemical groups and points of attachment on various dyes can be found,for example, in U.S. Pat. Nos. 5,654,442 and 5,188,934 (Menchen et al.),6,020,481 (Benson et al.), 5,800,996 (Lee et al.), 6,025,505 (Lee etal.), 5,821,356 (Khan et al.), 5,770,716 (Khan et al.), 6,088,379(Benson et al.), 6,051,719 (Benson et al.), 6,096,875 (Khan et al.),6,080,868 (Lee et al.), U.S. patent application Ser. No. 09/325,243filed Jun. 3, 1999 (Lam et al.), 09/498,702 filed Feb. 7, 2000 (Upadhyaet al.), 09/564,417 filed May 2, 2000 (Menchen et al.), and Ser. No.09/433,093 filed Nov. 3, 1999 (Lee et al.). In one preferred embodiment,for xanthene derivatives that contain a C9 phenyl group, such as arhodamine dye or fluorescein dye, the linker is attached to the dye viaa 5-carboxyphenyl (para to the xanthene C9 carbon atom) or6-carboxyphenyl group (meta to the xanthene C9 carbon atom). In anotherpreferred embodiment, for xanthene dyes generally, the linker ispreferably attached to a 4-carbon atom or 5-carbon atom on the xanthenering. In a third preferred embodiment, for rhodamine-type xanthene dyesand rhodamine dyes, the linker is attached to the 3 or 6-nitrogen atomof the xanthene ring. Further guidance for forming conjugates of theinvention can be found below with reference to the Examples herein.

The dye-labeled nucleobase of the invention may also have the formB-L1-D1-L2-D2, wherein B is a nucleobase, L1 and L2 are linkers suchthat at least one of L1 and L2 is an anionic linker, and D1 and D2 aremembers of a fluorescent donor/acceptor pair. In one embodiment, D1 is adonor dye, and D2 is an acceptor dye. In another embodiment, D2 is adonor dye, and D1 is an acceptor dye. For donor/acceptor pairs, it isunderstood that the donor dye and acceptor dye have different(non-identical) spectral properties. Thus, although the donor andacceptor may have the same type of aromatic ring structure (e.g., whenboth the donor and acceptor are fluorescein dyes, or both are rhodaminedyes), different spectral properties can arise for the donor andacceptor due to the nature of the substituents on each one. The donordye is effective to enhance the intensity of fluorescence emission ofthe acceptor dye relative to the intensity that would be observed in theabsence of the donor dye under the same conditions. Conjugates of thisform may be referred to herein as “FRET probes”, “FRET-labeledconjugates” or FRET-labeled nucleotides because upon excitation of thedonor dye, the conjugate can undergo nonradiative fluorescence resonanceenergy transfer from the donor to the acceptor, such that the acceptordye can then emit fluorescent light at a second wavelength in responsethereto.

The donor dye and acceptor dye can be any fluorescent dye, and are eachpreferably fluorescent aromatic dyes. For example, the donor andacceptor dye, taken separately, can be a xanthene, rhodamine,dibenzorhodamine, fluorescein, [8,9]benzophenoxazine, cyanine,phthalocyanine, squaraine, or bodipy dye. Furthermore, the donor andacceptor dyes can be linked together using any of a variety ofattachment sites on each dye. For example, if D1 is a fluorescein and D2is a rhodamine (both of which contain pendent phenyl groups attached toC9 of the xanthene rings), D1 can be linked via its xanthene ring(preferably via C4)) to the pendent phenyl ring of D2 (e.g., via a 5- or6-carboxy group on the pendent phenyl group). This is referred to as ahead to tail arrangement. Alternatively, the positions of theconnections can be reversed, such that D2 is linked via its xanthenering to the pendent phenyl ring of D1 (another example of a head to tailarrangement). In other alternatives, D1 and D2 can be connected tail totail, via their pendent phenyl rings, or head to head, via theirxanthene rings, for example.

As noted above, at least one of L1 and L2 is an anionic linker. Theproperties of such anionic linkers are generally as discussed foranionic linker L above.

In one embodiment, L1 is an anionic linker and L2 is a non-anioniclinker. For L2, any of a variety of non-anionic linkers can be used toconnect D1 to D2. General considerations for forming donor-acceptorconjugates are discussed in U.S. Pat. Nos. 5,863,727, 5,800,996,5,945,526, and 6,008,379, for example. In one set of embodiments,D1-L2-D2 may comprise one of structures (a), (b) or (c) below:

-   -   (a) -D1-R₂₁Z₁C(O)R₂₂R₂₈-D2    -   (b) -D1-R₂₈R₂₂C(O)Z₁R₂₁-D2    -   (c) -D1-R₂₈R₂₂R₂₈-D2        wherein: R₂₁ is C₁-C₅ alkyldiyl, Z₁ is NH, S, or O, R₂₂ is an        alkene, diene, alkyne, or a 5- or 6-membered ring having at        least one unsaturated bond or a fused ring structure, and R₂₈ is        a bond or spacer group. Details and examples of such inter-dye        linkers can be found in U.S. Pat. No. 5,800,996, for example. In        certain embodiments, R₂₂ is ethenediyl, ethynediyl,        1,3-butadienediyl, or 1,3-butadiynediyl.

In another embodiment, L1 is a non-anionic linker and L2 is an anioniclinker. In this case, any of a variety of non-anionic linkers can beused to connect B to D1. Descriptions of exemplary nonanionic linkerscan be found in the references in Table 1 above. For example, L1 can beor contain any of the following non-limiting examples:

-   -   —C≡CCH₂NH-—    -   —C≡CCH₂OCH₂CH₂NH—    -   —C≡CCH₂OCH₂CH₂OCH₂CH₂NH—    -   —C≡CCH₂NHC(O)(CH₂)₅NH—    -   —C≡CC(O)NH(CH₂)₅NH—    -   —C═CHC(O)NH(CH₂)₅NH—    -   —C≡C—(p-C₄H₆)OCH₂CH₂NH—    -   —C≡C—(p-C₆H₄)OCH₂CH₂NH—    -   —C≡C—(p-C₆H₄)-(p-C₆H₄)—C≡C—    -   —C≡C—(p-C₄H₆)—    -   —C≡C—C≡C—        wherein the left-hand ethene or ethyne moiety is linked to the        nucleobase, and the right hand bond is typically linked directly        to the dye or is linked indirectly to the dye through a carbonyl        group. Additional nonanionic linkers are shown in the exemplary        compounds in the attached Figures.

More generally, non-anionic linkers encompass linkers that are eithercharge-neutral or are positively charged. Charge-neutral linkers referto linkers that either contain no charged groups at pH 7 (i.e., has nocharged group having a pKa between 6 and 8), or contain equal numbers ofpositively and negatively charged groups which cancel to provide a netcharge of zero. A positively charged linker is positively charged at pH7, e.g., due to the presence of an ammonium ion or imidazole ion, forexample. Preferably, a non-anionic linker is a charge-neutral linker.Preferably, the charge-neutral linker contains no charged groups at pH7.

When L1 and L2 are both anionic linkers, the structures of L1 and L2 canbe the same or different, and the anionic group(s) in the linkers canalso be the same or different. Further guidance regarding conjugatestructures in accordance with the invention is provided below.

Compounds of the invention may be prepared by any suitable syntheticmethod. Typically, conjugates of the invention are formed using amodular approach in which a nucleobase (which may be provided in theform of a nucleoside or nucleotide containing the nucleobase, forexample), a first dye, a second dye (if present), and one or morelinkers or linker precursors, are combined in serial and/or parallelsteps to produce the desired labeled product. Several exemplaryapproaches are illustrated in the Examples below, which describesyntheses of several different dye-labeled nucleotides containinglinkers of various lengths and compositions.

Example 1 describes a synthetic method for preparing a labelednucleotide in accordance with the invention, which contains a (i) firstdye linked via C-8 of a 7-deazaadenine nucleobase by an anionic linkerthat contains a phosphate diester moiety within the chain of linkeratoms, and (ii) a second dye linked to the first dye by a charge-neutral(non-anionic) linker. The linker between the nucleobase and the firstdye contains 13 linker chain atoms. The linker between the first andsecond dyes contains 10 linker atoms. In this example, a bifunctionallinker moiety 7 is formed in several steps by first reacting a cyclicphosphoramidite 1 with methyl glycolate 2, followed by oxidation, toform phosphate compound 3. After removal of a methyl group from thephosphate to produce 4, and deprotection of the amino and carboxylgroups to produce compound 5, an Fmoc protecting group is attached tothe amine to produce carboxylic acid 6. Activation of the carboxylicacid with N-hydroxysuccinimide (NHS) produces ester 7, which is aversatile linker synthon. Ester 7 is then reacted with7-aminopropargyl-7-deazaadenosine triphosphate 8 to form compound 9. Forreaction with compound 9, dye compound 11 was prepared by reactingp-aminomethylbenzoic acid with Fmoc acid chloride to form Fmoc protectedp-aminomethylbenzoic acid. After activation of the benzoic acid withNHS, the NHS ester product was reacted with4′-aminomethyl-6-carboxyfluorescein to produce the expected amideadduct. The 6-carboxyl group was then reacted with NHS to produce dyecompound 11. Reaction of compounds 10 and 11 produced adduct 12, inwhich an anionic (phosphate-containing) linker is formed fully betweenthe fluorescein dye and the nucleobase. After removal of the Fmoc group,the resultant free amine compound 13 was reacted with rhodamine NHSester 14 to form dye-labeled nucleotide 15.

An alternative method for preparing compound 10 used in Example 1 isprovided in Example 2. This Example describes a synthetic approach inwhich a linker synthon 19 containing a phosphate monoester is preparedfor reaction with a nucleoside containing an iodinated nucleobase(7-iodo-7-deazaadenine). In brief, 3-amino-1-propyne 16 is reacted withmethyl glycolate 2 to form the expected amide product 17. Reaction of 17with cyclic phosphoramidite 1 followed by oxidation affords phosphatetriester 18. After removal of the phosphate group, resultantphosphodiester compound 19 is reacted with iodo-nucleoside 20 to affordadduct 21. The 5′-hydroxyl group of the nucleoside can be converted to atriphosphate group by reaction with phosphorous oxychloride to formdichloromonophosphate 22, followed by addition of pyrophosphate toprovide nucleoside triphosphate 23. Removal of the trifluoracetylprotecting group from the terminal amine nitrogen provides synthon 10.

Example 4 describes synthesis of a synthetic method for preparing aFRET-labeled nucleotide which contains a (i) first dye linked to a7-deazaadenine nucleobase by a charge-neutral (non-anionic) linker, and(ii) a second dye linked to the first dye by an anionic linker thatcontains a monoanionic sulfonic acid moiety. The linker between thenucleobase and the first dye contains 5 linker chain atoms. The linkerbetween the first and second dyes contains 10 linker atoms. As detailedin Example 4, p-aminomethylbenzoic acid 24 is reacted with sulfuric acidto form the meta-sulfonated product 25. Reaction with Fmoc-succinimideaffords Fmoc protected amine 26, which is then reacted withN-hydroxysuccinimide to form NHS ester 27. For reaction with NHS ester27, dye-labeled nucleoside triphosphate 30 can be prepared by reactingaminopropargyl nucleoside triphosphate 8 with dye intermediate 28 (afluorescein dye containing a trifluoroacetyl-protected 4′-aminomethylgroup and an NHS ester of a 6-carboxyl group) to afford dye-labelednucleotide 29, followed by removal of the trifluoroacetyl (TFA) group toafford amine compound 30. Reaction of NHS ester 27 and compound 30affords Fmoc-protected compound 31. After removal of the Fmoc group,resultant amine compound 32 is reacted with dye NHS ester 14 to afforddye-labeled nucleotide 33. It can be seen that the linker between thefirst and second dyes contains a sulfonic acid group which is attachedto a benzene moiety in the linker chain.

In Example 5, a protocol is described for preparing a labeled nucleotidein which first and second dyes of a donor-acceptor pair are linked by aphosphate-containing linker, and the donor dye is linked to a nucleobaseby a charge-neutral linker. This nucleotide product differs from theproduct of Example 1 since the anionic phosphate linker is locatedbetween the first dye and the nucleobase. The product of Example 5 alsodiffers from the product of Example 4 since the anionic linker betweenthe two dyes contains a phosphate monoester in the linker chain, ratherthan a sulfonic acid group attached to the linker chain. The compoundsalso differ in the lengths of some of the linkers.

As detailed in Example 5, aminopropargyl nucleotide 8 is combined withdye NHS ester 11 to afford Fmoc-protected compound 34. Following removalof the Fmoc group, resultant amine 35 is reacted with ester 7 to produceFmoc protected dye-labeled nucleotide 36. After removal of the Fmocgroup, resultant amine 37 is reacted with dye NHS ester 14 to afforddye-labeled nucleotide 38.

A method for preparing a FRET-labeled nucleotide containing two anioniclinkers is described in Example 6. In particular, the anionic linkerbetween the nucleobase and first dye contains a phosphate diestermoiety, and the anionic linker between the first and second dyescontains a sulfonic acid moiety. In the method described in Example 6,nucleotide amine 10 (Examples 1 and 2) is reacted with dye NHS ester 28to form dye-labeled nucleotide 39, in which the dye and nucleotide arelinked by a phosphate-containing linker. Removal of the Fmoc groupproduces amine compound 40, which is reacted with sulfonate-containingNHS ester 27 to afford Fmoc-protected compound 41. After removal of theFmoc group, resultant amine 42 is reacted with dye NHS ester 14 toafford dye-labeled nucleotide 43.

Example 7 illustrates how the dye-labeled nucleotide compound 40 fromExample 6 can be used to form a conjugate of the invention by analternative route, relative to the route described in Example 1. InExample 1, the main linker synthon between the first and second dyes isprovided as part of compound 11, which contains a first dye (forattachment to the nucleobase) and an Fmoc-protected linker synthon thatis attached to the first dye. In the method of Example 7, the linker isprovided as part of a compound (44) which contains a linker synthonattached to the second dye. This compound can be reacted withdye-labeled nucleotide 40 to obtain desired product 15. Thus, Example 7illustrates how the order of connection of various synthons can bevaried, if desired, to synthesize a particular compound of theinvention.

Examples 8A-8B describe methods for preparing two FRET-labelednucleotides that are similar to the product of Example 1, except thatthe nucleobase is cytosine. In Example 8A, the linkers are the same asfor the product of Example 1. In Example 8B, the linker between thenucleobase and first dye is longer than the corresponding linker inExample 8A, due to the inclusion of an ethoxy group inserted after thepropargyl group.

Examples 9, 10 and 11 describe methods for making additionalFRET-labeled nucleobases (thymine) which contain selected anioniclinkers between first and second dyes. The method in Example 9 producesa product 64 that has a sulfonate-containing anionic linker between thetwo dyes, and a charge-neutral linker between the first dye andnucleobase. Product 69 from Example 10 has a phosphate-containinganionic linker between the first and second dyes. This anionic linker isalso longer than the linker of Example 9 (18 linker chain atoms versus10 linker chain atoms). The method in Example 11 produces a product 77similar to product 69, except that the nucleobase is 7-deazaguanosine,and the attached dye is different.

Methods for preparing several exemplary dye-labeled conjugates of theform B-L-D are provided in Examples 12, 13 and 18. Example 12A describesa method for preparing a dye-labeled conjugate 82 having an anioniclinker that contains a sulfonate group, as illustrated by a sulfonatedbenzene moiety. In Example 12B, dye-labeled conjugate 85 has a dianioniclinker (net formal charge of −2) that contains both a sulfonate groupand a phosphate group. In Example 13, dye-labeled conjugate 95 has ananionic linker containing a phosphodiester moiety within the linkerchain.

Example 18 (see also FIGS. 22A and 22B) provides a method of forming aconjugate of the form B-L-D having a linker that comprises a carboxylanionic group, as illustrated with a carboxy benzene moiety as part ofthe linker. It will be appreciated that carboxylic acid groups can alsobe included in linkers in other ways, by preparing appropriatecarboxylated linker synthons.

Examples 14, 15 and 16 provide additional methods for preparingFRET-labeled conjugates wherein the nucleobase and first dye are linkedby an anionic linker, and the donor and acceptor dyes are linked by acharge-neutral linker. The nucleobase in Examples 14 and 16 is7-deazaadenine. The nucleobase in Example 15 is 7-deazaguanine. InExample 14, a phosphodiester moiety is linked to a 7-propargyl group onthe nucleobase, and the remainder of the linker is provided as anethylaminoacyl moiety linked to the pendent phenyl ring of a fluoresceindye. In Example 15, a phosphodiester moiety is linked to the nucleobaseby a 7-propargylphenylpropynyl group, and the remainder of the linker isprovided as an ethylaminoacyl moiety. In Example 16, a phosphonatemonoester is linked to the nucleobase by a methylacylaminopropargylgroup, and the remainder of the linker is provided as an ethylaminoacylmoiety.

The foregoing examples illustrate a broad variety of dye-labelednucleobase compounds in accordance with the invention, including fourdifferent types of nucleobases, linkers of different compositions andlengths, a number of different types of dyes, and various combinationsthereof. Several specific examples of conjugates of the formB-L1-D1-L2-D2 are described wherein L1, L2 or both, are anionic linkersof various types and lengths. Specific examples of conjugates of theform B-L-D are also provided (Examples 12A, 12B, and 13), andmodifications should be immediately apparent from the FRET pair Examplesin which L1 is an anionic linker.

The present invention also includes nucleosides and nucleotidescontaining conjugates in accordance with the invention. Particularlypreferred nucleosides/tides of the present invention are shown below inthe following formula:

wherein W₁ is OH, H, F, Cl, NH₂, N₃, or OR, where R is C1-C6 allyl(e.g., OCH₃ or OCH₂CH₃); W₂ is OH or a group capable of blockingpolymerase-mediated template-directed primer extension (such as H, F,Cl, NH₂, N₃, or OR, where R is C1-C6 alkyl (e.g., OCH₃ or OCH₂CH₃)); W₃is OH, or mono-, di- or triphosphate or a phosphate analog thereof; andLB (labeled base) represents a dye-labeled nucleobase conjugate of theinvention. In one embodiment, W₁ is not OH. In another embodiment, W₂ isnot OH, so that the compound is not 3′ extendable. In anotherembodiment, W₁ and W₂ are selected from H, F, and NH₂. In furtherembodiments, W₁ is F and W₂ is H, or W₁ is H and W₂ is F, or W₁ and W₂are each F, or W₁ and W₂ are each H. In addition, for each of theforegoing embodiments for W₁ alone, W₂ alone, and W₁ and W₂ incombination, it is contemplated that W₃ can be OH, monophosphate,diphosphate, or triphosphate. For LB, exemplary nucleobases includeadenine, 7-deazaadenine, 7-deaza-8-azaadenine, cytosine, guanine,7-deazaguanine, 7-deaza-8-azaguanine, thymine, uracil, and inosine.

For example, in one particular embodiment, when W₃ is triphosphate, thepresent invention includes nucleotide triphosphates having the structureshown in the formula below:

wherein X is H or F. Such terminator nucleotides, and others discussedabove which lack a 3′ OH group, find particular application as chainterminating agents in Sanger-type DNA sequencing methods utilizingfluorescent detection, and also in minisequencing.

In another embodiment, the invention includes deoxynucleotidetriphosphates having the structure shown in the formula below:

wherein LB is defined as above. Such compounds are examples of 3′extendable nucleotides. Labeled 2′-deoxynucleotides of this type findparticular application as reagents for labeling polymerase extensionproducts, e.g., in the polymerase chain reaction and nick-translation.

In yet another embodiment, the invention includes ribonucleotidetriphosphates having the structure shown in the formula below:

wherein LB is defined as above. Labeled nucleotides of this type findparticular application as reagents for and in sequencing methods thatutilize labile nucleotides having cleavable internucleotide linkages, asdiscussed for example in U.S. Pat. No. 5,939,292 (Gelfand et al.),Eckstein, Nucl. Acids Res. 16:9947-9959 (1988), and Shaw, Nucl. AcidsRes. 23:4495 (1995).

The invention also provides polynucleotides and mixtures ofpolynucleotides that contain one or more different nucleobase-dyeconjugates of the type discussed above. Such polynucleotides are usefulin a number of important contexts, such as DNA sequencing, ligationassays, the polymerase chain reaction (PCR), probe hybridization assays,and various other sequence detection or quantitation methods.

Dye-containing polynucleotides (also referred to herein as labeledpolynucleotides) may be synthesized enzymatically, e.g., using a DNA orRNA polymerase, nucleotidyl transferase, ligase, or other enzymes, e.g.,Stryer, Biochemistry, Chapter 24, W.H. Freeman and Company (1981), or bychemical synthesis, e.g., by the phosphoramidite method, thephosphite-triester method, or the like. Dye-labels of the invention maybe introduced during enzymatic synthesis utilizing labeled nucleotidetriphosphate monomers as described above, or during chemical synthesisusing labeled non-nucleoside or nucleoside phosphoramidites, or may beintroduced subsequent to synthesis. Exemplary methods for forminglabeled polynucleotides can be found in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, NY (1989), U.S. Pat. No. 6,008,379 (Benson et al.), andreferences cited therein.

Generally, if a labeled polynucleotide is made using enzymaticsynthesis, the following procedure may be used. An oligonucleotideprimer is annealed to a complementary sequence in a template DNA strand.A mixture of deoxynucleotide triphosphates (such as dGTP, dATP, dCTP,and dTTP) is added, where at least one of the deoxynucleotides containsa nucleobase-dye conjugate of the invention. In the presence of apolymerase enzyme, a dye-labeled polynucleotide is formed byincorporation of a labeled deoxynucleotide during polymerase-mediatedstrand synthesis. In an alternative enzymatic synthesis method, twoprimers are used instead of one, one primer complementary to the +strandand the other complementary to the −strand of the target, the polymeraseis a thermostable polymerase, and the reaction temperature is cycledbetween a denaturation temperature and an extension temperature, therebyexponentially synthesizing (amplifying) a labeled complement to thetarget sequence by PCR, e.g., PCR Protocols, Innis et al. eds., AcademicPress (1990).

Labeled polynucleotides may be chemically synthesized using any suitablemethod, such as the phosphoramidite method. Detailed descriptions of thechemistry used to form polynucleotides by the phosphoramidite method areprovided elsewhere, e.g., Caruthers et al., U.S. Pat. Nos. 4,458,066 and4,415,732, Caruthers et al., Genetic Engineering 4: 1-17 (1982), UsersManual Model 392 and 394 DNA/RNA Synthesizers, pages 6-1 through 6-22,Applied Biosystems, Part No. 901237 (1991). Descriptions of thephosphoramidite method and other synthesis methods for makingpolynucleotides containing standard phosphodiester linkages or linkageanalogs can be found in Gait, Oligonucleotide Synthesis, IRL Press(1990), and S. Agrawal, Protocols for Oligonucleotides and Analogs,Methods in Molecular Biology Vol. 20, Humana Press, Totowa, N.J. (1993).

The phosphoramidite method is a preferred method because of itsefficient and rapid coupling and the stability of the startingmaterials. The synthesis is performed with a growing polynucleotidechain attached to a solid support, so that excess reagents, which are inthe liquid phase, can be easily removed by filtration, therebyeliminating the need for purification steps between synthesis cycles.

III. METHODS

The nucleobase-dye conjugates of the present invention are suited forany method utilizing fluorescent detection, particularly methodsrequiring simultaneous detection of analytes which are not wellseparated by electrophoresis. The present invention is particularly wellsuited for detecting classes of polynucleotides that have been subjectedto a biochemical separation procedure, such as electrophoresis.

In one aspect, the invention provides a method of identifying one ormore polynucleotide(s). The method utilizes one or more labeleddifferent-sequence polynucleotides, which may have the same lengths ordifferent lengths, wherein each different-sequence polynucleotidecontains a unique nucleobase-dye conjugate. The one or more labeleddifferent-sequence polynucleotides are separated by electrophoresis toseparate different-sequence polynucleotides on the basis of size. Eachdifferent-sequence polynucleotide can then be identified on the basis ofits electrophoretic mobility and fluorescence signal.

The polynucleotide(s) may be formed by any appropriate method, with theproviso that each different polynucleotide is identifiable on the basisof a unique combination of electrophoretic mobility and fluorescencesignal. For example, two different polynucleotides may contain identicaldye moieties but may exhibit different electrophoretic mobilities.Alternatively, two different polynucleotides can contain different dyemoieties that produce distinct (spectrally resolvable) fluorescencesignals but can exhibit the same electrophoretic mobilities. In anotherexample, different polynucleotides can differ in both their fluorescencesignals and mobilities.

In one embodiment, the method can be used in a multiplex format in whichdifferent labeled polynucleotides are formed by reaction with (i) aplurality of different target sequences and (ii) a plurality ofdifferent polynucleotides that are complementary to the targetsequences. For example, the different polynucleotide can be designed toundergo a change in structure after hybridization to their complementarytarget sequences in a polynucleotide sample, e.g., due to modificationby enzyme action, thereby producing different labeled polynucleotideshaving unique combinations of mobility and fluorescence to allowidentification. Such reactions can be performed simultaneously in asingle reaction mixture or can be performed in separate reactionmixtures that can be combined prior to electrophoretic separation.Several exemplary assay formats for producing such labeledpolynucleotides are discussed below.

Sanger-type sequencing involves the synthesis of a DNA strand by a DNApolymerase in vitro using a single-stranded or double-stranded DNAtemplate whose sequence is to be determined or confirmed. Synthesis isinitiated at a defined site based on where an oligonucleotide primeranneals to the template. The synthesis reaction is terminated byincorporation of a nucleotide analog that will not support continued DNAelongation. Exemplary chain-terminating nucleotide analogs include the2′,3′-dideoxynucleoside 5′-triphosphates (ddNTPs) which lack the 3′-OHgroup necessary for 5′ to 3′ DNA chain elongation. When properproportions of dNTPs (2′-deoxynucleoside 5′-triphosphates) and one ofthe four ddNTPs are used, enzyme-catalyzed polymerization will beterminated in a fraction of the population of chains at each site wherethe ddNTP is incorporated. If labeled ddNTPs are used for each reaction,a desired sequence read can be obtained by detection of the fluorescencesignals of the terminated chains during or after separation byhigh-resolution electrophoresis. In the chain termination method, dyesof the invention can be attached to either sequencing primers orterminator nucleotides.

In “fragment analysis” or “genetic analysis” methods, labeledpolynucleotide fragments can be generated through template-directedenzymatic synthesis using labeled primers or nucleotides, e.g., bypolynucleotide ligation or polymerase-directed primer extension. Theresultant fragments are then subjected to a size-dependent separationprocess, e.g., electrophoresis or chromatography, and the separatedfragments are detected, e.g., by laser-induced fluorescence. In aparticular embodiment, multiple classes of polynucleotides are separatedsimultaneously and the different classes are distinguished by spectrallyresolvable labels.

A fragment analysis method, known as amplified fragment lengthpolymorphisim detection (AmpFLP), is based on amplified fragment lengthpolymorphisms, i.e., restriction fragment length polymorphisms that areamplified by PCR. These amplified fragments of varying size serve aslinked markers for following mutant genes in family lineages. The closerthe amplified fragment is to the mutant gene on the chromosome, thehigher the linkage correlation. Because genes for many genetic disordershave not been identified, these linkage markers serve to help evaluatedisease risk or paternity. In the AmpFLP technique, the polynucleotidesmay be labeled by using a labeled polynucleotide PCR primer, or byutilizing labeled nucleotide triphosphates in the PCR.

In another fragment analysis method, known as nick translation, one ormore unlabeled nucleotide subunits in a double-stranded DNA molecule arereplaced with labeled subunits. Free 3′-hydroxyl groups are createdwithin the unlabeled DNA by “nicks” caused by treatment withdeoxyribonuclease I (DNAase I). The DNA polymerase I then catalyzes theaddition of one or more labeled nucleotides to the 3′-hydroxyl of thenick. At the same time, the 5′ to 3′-exonuclease activity of this enzymecan remove one or more nucleotide subunits from the 5′-phosphorylterminus of the nick. A new nucleotide with a free 3′-OH group isincorporated at the position of the excised nucleotide, and the nick isshifted along by one nucleotide unit in the 3′ direction. This 3′ shiftwill result in the sequential addition of new labeled nucleotides to theDNA with the removal of existing unlabeled nucleotides. Thenick-translated polynucleotide is then analyzed using a separationprocess, e.g., electrophoresis.

Another exemplary fragment analysis method is based on the variablenumber of tandem repeats, or VNTRs. VNTRs are regions of double-strandedDNA that contain adjacent multiple copies of a particular sequence, withthe number of repeating units being variable among different members ofa population (e.g., of humans). Examples of VNTR loci are pYNZ22,pMCT118, and Apo B. A subset of VNTR methods are based on the detectionof microsatellite repeats, or short tandem repeats (STRs), i.e., tandemrepeats of DNA characterized by a short (2-4 bases) repeated sequence.One of the most abundant interspersed repetitive DNA families in humansis the (dC-dA)n-(dG-dT)n dinucleotide repeat family (also called the(CA)n dinucleotide repeat family). There are thought to be as many as50,000 to 100,000 (CA)n repeat regions in the human genome, typicallywith 15-30 repeats per block. Many of these repeat regions arepolymorphic in length and can therefore serve as useful genetic markers.Preferably, in VNTR or STR methods, label is introduced into thepolynucleotide fragments by using a dye-labeled PCR primer.

In another example, known sometimes as an oligonucleotide ligation assay(OLA), two polynucleotides (probe pair) which are complementary toadjacent regions in a target sequence are hybridized to the targetregion of a polynucleotide, to create a nicked duplex structure in whichthe ends of the two polynucleotide abut each other. When the ends of thehybridized polynucleotide probes match (basepair with) correspondingsubunits in the target, the two probes can be joined by ligation, e.g.,by treatment with ligase. The ligated product is then detected,evidencing the presence of the target sequence. In a modification ofthis approach, known as the ligation chain reaction (or ligationamplification reaction), the ligation product acts as a template for asecond pair of polynucleotide probes which are complementary to theligated product from the first pair. With continued cycles ofdenaturation, reannealing and ligation in the presence of the twocomplementary pairs of probe, the target sequence is amplifiedexponentially, allowing very small amounts of target sequence to bedetected and/or amplified. Exemplary conditions for carrying out suchprocesses, including chemical ligation formats, are described in U.S.Pat. Nos. 5,962,223 (Whiteley et al.), 4,988,617 (Landegren et al.), and5,476,930 (Letsinger et al.), and European Patent Publications EP246864A (Carr et al.), EP 336731A (Wallace), and EP 324616A (Royer etal.).

Conveniently, a fragment analysis method such as any of those discussedabove can be performed in a multi-probe format, in which a sample isreacted with a plurality of different polynucleotide probes or probesets which are each specific for a different target sequence, such asdifferent alleles of a genetic locus and/or different loci. The probesare designed to have a unique combination of mobility and fluorescencesignal, to permit specific detection of the individual probes or probeproducts that are generated in the assay as a result of the presence ofthe different target sequences.

In the above fragment analysis methods, labeled polynucleotides arepreferably separated by electrophoretic procedures. Methods forelectrophoresis of nucleic acids are well known and are described, forexample in Rickwood and Hames, Eds., Gel Electrophoresis of NucleicAcids: A Practical Approach, IRL Press Limited, London (1981), Osterman,Methods of Protein and Nucleic Acid Research, Vol. 1 Springer-Verlag,Berlin (1984), Sambrook et al. (1989, supra), P. D. Grossman and J. C.Colburn, Capillary Electrophoresis: Theory and Practice, Academic Press,Inc., NY (1992), and U.S. Pat. Nos. 5,374,527, 5,624,800 and/or5,552,028. Typically, the electrophoretic matrix contains crosslinked oruncrosslinked polyacrylamide having a concentration (weight to volume)of between about 2-20 weight percent, and often about 4 to 8 percent.For DNA sequencing, the electrophoresis matrix usually includes adenaturing agent such as urea, formamide, or the like. Detailedexemplary procedures for forming such matrices are given by Maniatis etal., “Fractionation of Low Molecular Weight DNA and RNA inPolyacrylamide Gels Containing 98% Formamide or 7 M Urea,” in Methods inEnzymology, 65: 299-305 (1980), Sambrook et al. (1989, supra), and ABIPRISM™ 377 DNA Sequencer User's Manual, Rev. A, January 1995, Chapter 2(p/n 903433), Applied Biosystems, Foster City, Calif.). A variety ofsuitable electrophoresis media are also commercially available fromApplied Biosystems and other vendors, including non-crosslinked media,for use with automated instruments such as the Applied Biosystems “3700”and “3100” Instruments, by way of example. Optimal electrophoresisconditions, e.g., polymer concentration, pH, temperature, voltage,concentration of denaturing agent, employed in a particular separationdepends on many factors, including the size range of the nucleic acidsto be separated, their base compositions, whether they are singlestranded or double stranded, and the nature of the polynucleotides forwhich information is sought by electrophoresis. Accordingly applicationof the invention may require standard preliminary testing to optimizeconditions for particular separations.

During or after electrophoretic separation, the labeled polynucleotidescan be detected or identified by recording fluorescence signals andmigration times (or migration distances) of the separatedpolynucleotides, or by constructing a chart of relative fluorescent andorder of migration of the polynucleotides (e.g., as anelectropherogram). To perform such detection, the labeledpolynucleotides can be illuminated by standard means, e.g. a highintensity mercury vapor lamp, a laser, or the like. Typically, thelabeled polynucleotides are illuminated by laser light generated by aHe—Ne gas laser or a solid-state diode laser. The fluorescence signalscan then be detected by a light-sensitive detector, e.g., aphotomultiplier tube, a charged-coupled device, or the like. Exemplaryelectrophoresis detection systems are described elsewhere, e.g., U.S.Pat. Nos. 5,543,026, 5,274,240, 4,879,012, 5,091,652 and 4,811,218.

FIGS. 19A-19B, 20A-20B, and 21A-21B show various results in support ofthe present invention. The results were obtained for sequencingfragments prepared by template-dependent primer extension in thepresence of selected dye-labeled terminators following the methodologydescribed in U.S. Pat. No. 6,096,875 (see Example 12, except that theprimers were not labeled). Primer extension reactions were performed inthe presence of a single type of terminator to generate a ladder ofextension fragments. The two extension reactions in each study differedonly in the linkers that were present between the nucleobase and thedye, or between a donor dye and acceptor dye. Specifically, the linkerin one reaction mixture (A) is non-anionic, whereas the linker in theother reaction mixture (B) is an exemplary anionic linker.

For FIGS. 19A and 19B, two primer extension reactions were performedusing 7-deaza analogs of dideoxyguanosine-5′-triphosphate (ddGTP). Bothterminators contained only one dye, which is a dibenzoxanthene dye ofthe type described in U.S. Pat. No. 5,936,087 at FIG. 14, wherein R₁ isCH₂-p-C₆H₄ and R₂ is propyldiyl, and wherein the C4 and C10 of the benzogroups are sulfonated. The dye is linked by the 12-nitrogen atom of thedibenzoxanthene ring structure to C7 of the 7-deazaguanine ring. ForFIG. 19A, the structure of the L linker isB—C≡CCH₂OCH₂CH₂NHC(O)-p-C₆H₄—CH₂-D, wherein B represents C7 of thenucleobase and D represents N12 of the dye. For FIG. 19B, the structureof the L linker isB—C≡CCH₂OCH₂CH₂NHC(O)CH₂OP(O)(OH)OCH₂CH₂—NH—C(O)-p-C₆H₄CH₂-D, wherein Band D are as just described.

An arrow in FIG. 19A points to a middle peak flanked on each side by aleft peak and a right peak. The spacing between the left and middlefragments appears to be about equal to the spacing between the middleand right fragments, suggesting that the target sequence contains threecytosine subunits (complementary to the ddG terminator) which areseparated from each other by an equal number of intervening subunits.However, based on the known sequence of the target template (pGEM), the3′-terminal subunits of the fragments corresponding to the left andmiddle peaks are known to be separated by three intervening subunits,whereas the 3′-terminal subunits of the fragments corresponding to themiddle and right peaks are separated by only one intervening subunit. Inother words, the middle peak is four subunits longer than the left peak,and the right peak is only two subunits longer than the middle peak.Thus, based on the observed profile in FIG. 19A, a user would have greatdifficulty in determining the correct target sequence. For example, ifprimer extension had been performed in the presence of four spectrallyresolvable terminator, the middle peak could co-migrate with a longer orshorter fragment containing a different terminator, thereby obscuringthe correct order of the fragments and rendering indeterminate thesequence information.

In contrast, the profile in FIG. 19B, which was obtained using adye-labeled nucleobase in accordance with the invention, has theexpected spacing between the three peaks. The separation between theleft and middle peaks is twice the separation between the middle andright peaks, consistent with length differences of four and twosubunits, respectively, between the left, middle, and right peaks.Clearly, this profile would allow a user to more easily determine thecorrect target sequence. These results demonstrate how the use of ananionic linker in a dye-labeled nucleobase can significantly improve thecorrelation between fragment length and electrophoretic mobility ofdye-labeled polynucleotides.

FIGS. 20A and 20B show electrophoretic profiles obtained using twodifferent 7-deaza analogs of dideoxyadenosine-5′-triphosphate (ddATP).In this study, both terminators contained a donor/acceptor dye pair inwhich D1 is a 6-carboxyfluorescein containing a 5′-amino methyl group,and D2 is the same as the 5-carboxy-4,7-dichlororhodamine dye used inExample 1 herein. In both conjugates, D1 is linked via the 4′aminomethyl group to C5 of the pendent phenyl ring of D2 using the L2 linkerfrom Example 1. For FIG. 20A, B and D1 were linked by the L1 linker fromExample 4 (—C≡CCH₂NHC(O)—). For FIG. 20B, B and D1 were linked by an L1linker of the form: B—C—CCH₂NHC(O)-p-C₆H₄(SO₃)CH₂NHC(O)-D1, wherein thesulfonate group on the benzene ring is ortho to the aminomethyl group.

The profile in the left-hand window of FIG. 20A contains a single peakthat is separated from a quartet of four peals on the right. From theapproximately equal spacing between adjacent peaks in the quartet, auser would probably conclude that the target sequence contains fourconsecutive thymidine subunits. However, such a conclusion would beerroneous, since the target sequence actually contains an adenosinesubunit between the third and fourth thymidine subunits. A profilehaving a significantly improved profile is shown in the left-hand windowof FIG. 20B. In particular, the third and fourth peaks in the quartetare separated by a distance consistent with a length difference of twosubunits for those peaks.

With reference to the right-hand windows of FIGS. 20A and 20B, theright-hand window of FIG. 20A shows a set of closely eluting peaks whichare numbered 1, 3, 5, 6, 8, and 10. Based on the known templatesequence, the correct terminator sequence reading is 5′-ACACAACATA-3′(SEQ ID NO:1) (corresponding to a template sequence of 3′-TGTGTTGTAT-5′)(SEQ ID NO:2). Unfortunately, peaks 5 and 6 in the right-hand window ofFIG. 20A are separated from each other by more than one peak interval,so that a user might conclude that the two peaks are separated by anintervening fragment corresponding to the presence of an interveningsubunit in the target sequence. (One “peak interval” refers to theaverage spacing between adjacent peaks differing in length by onenucleotide, for a selected region of an electropherogram.) Each peakcorresponds to a unique-sequence fragment of DNA.

A profile having significantly improved spacing is shown in theright-hand window of FIG. 20B. In this case, peaks 5 and 6 are separatedby about one peak interval, as expected for fragments that differ inlength by one nucleotide subunit. Overall, the improvement in spacingbetween peaks 1, 3, 5, 6, 8 and 10 allows more accurate determination ofthe target sequence.

Yet another example of the advantages of the invention is illustrated inFIGS. 21A and 21B. In this study, extension reactions were performedusing 7-deaza analogs of ddATP. Both terminators contained a fluoresceindye (compound 33 from U.S. Pat. No. 6,008,379) which is linked via C5 ofthe pendent phenyl ring to C7 of the 7-deazaadenine ring. For FIG. 21A,the structure of the L linker is B—C≡C-p-C₆H₄—C≡CCH₂OCH₂CH₂NHC(O)-D,wherein B represents C7 of the nucleobase and D represents C5 of thependent phenyl ring of the dye. For FIG. 21B, the structure of the Llinker is B—C≡C-p-C₆H₄—C≡CCH₂OCH₂CH₂NHC(O)—CH₂OP(O)(OH)OCH₂CH₂NHC(O)-D,wherein B and D are as just described.

FIG. 21A shows a profile containing four peaks consisting of a singleton the left, a singlet in the middle, and a doublet on the right. Fromthe known target sequence, the singlets should be separated from eachother by four peak intervals (due to the presence of three interveningnon-T subunits in the target sequence), the middle singlet and left-handpeak in the doublet should also be separated by four peak intervals, andthe two peaks in the doublet should be separated from each other by onepeak interval. The profile in FIG. 21A is problematic because the middlesinglet is separated from the left-hand member of the doublet byapproximately 3.5 peak intervals. In the profile shown in FIG. 21B,however, the spacing is more uniform, such that the middle singlet andleft-hand peak in the doublet are separated by four peak intervals.Thus, the target sequence can be determined more easily using thedye-labeled terminator of FIG. 21B in accordance with the invention.

The results above demonstrate how the use of an anionic linker in adye-labeled nucleobase can significantly improve the correlation betweenfragment length and electrophoretic mobility of dye-labeledpolynucleotides.

In conclusion, the present invention provides conjugate compounds inwhich a dye and nucleobase are linked by an anionic linker, or, in thecase of energy transfer dyes, one or more linkers located between a dyeand a nucleobase and/or between energy transfer dyes are anioniclinkers. Such linkers can be used in a variety of different forms andmay include any of a variety of different anionic groups, such thatbase-pairing characteristics of the nucleobase and fluorescentproperties of the dye are retained. Compounds of the invention areuseful in nucleoside and nucleotides that can be incorporated intopolynucleotides for detection. In particular, polynucleotides containingdye-labeled conjugates of the invention show reduced sequence-dependentvariations in electrophoretic mobility. Thus, the invention provideselectrophoretic separation patterns having more even spacing betweennearby polynucleotide bands, as well as reduced band-compression,resulting in a more consistent and uniform relationship betweenpolynucleotide length and electrophoretic mobility. Furthermore,nucleoside triphosphates that contain nucleobase-dye conjugates of theinvention are good substrates for polymerase enzymes which can be usedto incorporate such nucleotides into polynucleotides to form labeledpolynucleotides. This is advantageous in terminator-based sequencingmethods. When compounds of the invention are incorporated at the 3′ endsof sequencing fragments, artifacts in electrophoretic mobility arereduced, so that accuracy of base-calling can be improved.

IV. KITS

The invention also provides kits for performing the various methods ofthe invention. For nucleic acid sequencing, the kit comprises at leastone labeled nucleoside triphosphate comprising a conjugate describedherein. The kit may also include one or more of the followingcomponents: a 3′-extendable primer, a polymerase enzyme, one or more 3′extendable nucleotides which are not labeled with conjugate, and/or abuffering agent. In some embodiments, the kit includes at least onelabeled nucleoside triphosphate that is nonextendable. In otherembodiments, the kit comprises four different labeled nucleosidetriphosphates which are complementary to A, C, T and G, and each ofwhich contains a distinct conjugate as described herein. In yet anotherembodiment, the labeled nucleoside triphosphates are nonextendable. Inanother embodiment, the labeled nucleoside triphosphates are extendableribonucleoside triphosphates. In another embodiment, the kit comprisesat least one labeled, nonextendable nucleoside triphosphate comprising aconjugate described herein, and one or more of the following components:a 3′-extendable primer, a polymerase enzyme, and/or a buffering agent.

The operation of the invention can be further understood in light of thefollowing non-limiting examples which illustrate various aspects of theinvention.

EXAMPLES Materials and Methods

Unless indicated otherwise, all reagents and anhydrous solvents werepurchased from Aldrich Chemicals. Thin layer chromatography (TLC)analysis was conducted on aluminum plates precoated with 250 μm layersof silica gel 60-F254. Compounds were located by UV-VIS lamp and/or bycharring with aqueous K₂MnO₄. Flash column chromatography purificationwas carried out using EM Science silica gel 60 angstrom (230-400 MeshASTM). NMR spectra were recorded in deuterated solvents (CDCl₃, CH₃OD,and D₂O with an internal Me₄Si standard, δ 0). ¹H NMR spectra wererecorded at 300 MHz, ¹³C NMR spectra at 75.7 MHz, ¹⁹F NMR spectra at282.23 MHz, and ³¹ P NMR spectra at 121.44 MHz. In all cases, theobserved NMR spectra were in agreement with the indicated structures.Satisfactory mass spectra were also obtained for the reported compounds.

Anion-exchange high-performance chromatography (AE-HPLC) was performedas follows. Column: Aquapore™ AX300, 7 μm particle size and 220×4.6 mm(PE Applied Biosystems). Gradient: 40% acetonitrile: 60%triethylammonium bicarbonate (TEAB, 0.1 M) to 40% acetonitrile: 60% TEAB(1.5 M) at 1.5 ml/min over 20 minutes. Detection: UV absorbance at 260nm or λmax of each dye compound.

Reverse phase high-performance chromatography (RP HPLC) was performed asfollows. Column: Spheri-5 RP-C18, 5 μm particle size, 220×4.6 mm (PEApplied Biosystems); gradient: 95% triethylammonium acetate (TEAA, 0.1M): 5% acetonitrile to 50% acetonitrile: 50% TEAA at 1.5 ml/min over 20minutes and then to 100% acetonitrile over 5 minutes.

Example 1 Synthesis of Dye-Nucleotide Conjugate 15

Methyl glycolate 2 (4.5 eq.) was added to Amino-Link™ 1 (1 eq.)(Connell, C., et al., BioTechniques 5:342-348 (1987); U.S. Pat. No.4,757,151), followed by 4-N,N-dimethyl aminopyridine (DMAP) (0.1 eq.).The mixture was stirred at ambient temperature for 1 hour. After thereaction was complete (TLC analysis), the solution was cooled withice-bath and then treated with a solution of 3-chloroperoxybenzoic acid(4 eq.) in methylene chloride. The ice-bath was removed. After 30minutes, an aqueous solution of NaHSO₃ (10%) was added. The mixture wasdiluted with ethyl acetate. The organic layer was washed with NaHSO₃(10%), saturated solution of NaHCO₃, and dried with Na₂SO₄. The crudeproduct was purified by flash chromatography to afford compound 3.

To a solution of compound 3 (36 mM, 1 eq.) in methylethylketone wasadded NaI (10 eq.). The mixture solution was heated at reflux for 3hours. Solvent was removed under vacuum to afford crude compound 4 withNaI which was used as such without further purification.

Crude compound 4 (1 eq.) was dissolved in 0.3 M solution of LiOH (5 eq.)in a mixed solvent H₂O:CH₃OH (1:3). The mixture was stirred overnight.Solvent was removed to afford crude compound 5 which was then dissolvedin aqueous Na₂CO₃ (5%). N-(9-Fluorenylmethoxy-carbonyloxy)succinimide(FmocOSu, 1.5 eq.) in THF was added in one portion. The mixture wasstirred at ambient temperature for 3 hours. The crude product waspurified by flash chromatography to afford compound 6.

Compound 6 (1 eq.) was dissolved in ethyl acetate and the resultantsolution was washed with aqueous solution of HCl (10%). The organiclayer was dried with Na₂SO₄. Concentration under vacuum gave a yellowoil which was dissolved in anhydrous CH₂Cl₂. N-hydroxysuccinimide (4eq.) was added. The solution was cooled with an ice-bath and thentreated with dicyclohexyl carbodiimide (DCC, 2 eq.). The ice-bath wasthen removed, and stirring was continued for 2 hours (with TLCanalysis). When the reaction was complete, ethyl acetate was added andthe solution was washed with aqueous solution of HCl (5%). Removal ofsolvent gave compound 7.

Nucleoside triphosphate 8(7-(3-amino-1-propynyl)-2′,3′-dideoxy-7-deazaadenosine-5′-triphosphate—seeU.S. Pat. Nos. 5,047,519 and 5,151,507 by Hobbs et al. for synthesis) in100 mM TEA-bicarbonate solution (pH 7.0) was evaporated to dryness. Thedried residue was suspended in a solution of 250 mM bicarbonate (pH9.0). A solution of compound 7 in DMSO was added. After 1 hour, thereaction mixture was purified by HPLC (AX-300 anion exchange). Theproduct fractions were collected, concentrated to dryness, and purifiedby RP HPLC (C-18 reverse phase) to afford compound 9.

Ammonium hydroxide solution (28-30%) was added to dried compound 9. Thesolution was heated to 55° C. for 20 minutes. Concentration under vacuumgave crude compound 10 which was purified by HPLC (C-18 reverse phase).

NHS-ester dye intermediate 11 was prepared by reacting thefluorenylmethoxy-carbonyloxy ester of N-hydroxysuccinimide with the HClsalt of p-aminomethylbenzoic acid (both commercially available) in thepresence of base to form the expected N-Fmoc derivative. This productwas then reacted with N-hydroxysuccinimide in the presence of DCC toform the NHS ester of the benzoic acid carboxyl group. This NHS esterwas then reacted with 4′-aminomethyl-6-carboxyfluorescein (M. T.Shipchandler et al., Anal Biochem. 162:89-101 (1987)) to form theexpected product. This product was then reacted withN-hydroxysuccinimide in the presence of DCC to produce NHS-ester dyeintermediate 11.

Compound 10 was suspended in a solution of 250 mM bicarbonate (pH 9.0).Then a solution of dye intermediate 11 in DMSO was added. The reactionmixture was placed in the dark at ambient temperature for 2 hours.Purification was done by HPLC (AX-300 anion exchange). The recovereddye-labeled compound 12 was dried and then heated at 55° C. in ammoniumhydroxide (28-30%) for 20 minutes. Concentration under vacuum gave crudecompound 13 which was purified by HPLC (C-18 reverse phase).

Compound 13 was suspended in a solution of 250 mM bicarbonate (pH 9.0).Then a solution of dye 14 (e.g., see Example 17 or U.S. Pat. No.5,847,162 for synthesis) in DMSO was added. The reaction mixture wasplaced in dark at ambient temperature for 2 hours. Purification was doneby HPLC, AX-300 anion exchange and then C-18 reverse phase to affordpure nucleotide-dye compound 15, which contains a phosphate diestermoiety within the chain of linker atoms linking the inner dye to thenucleotide.

Example 2 Alternative Synthesis of Nucleotide 10

A solution of propargyl amine 16 (3-amino-1-propyne, 3.4 eq.), DMAP (0.1eq.), and methyl glycolate 2 (1 eq.) was heated to reflux for 2 hours.The reaction solution was cooled to ambient temperature and was pouredinto aqueous solution of HCl (10%). The solution was extracted withethyl acetate. Concentration under vacuum gave desired compound 17 as ayellow solid.

In a manner similar to the method described in Example 1, reaction ofcompound 17 (1 eq.) and Amino-Link™ 1 (1 eq.) followed by oxidation withmCPBA gave compound 18 which was purified by flash columnchromatography. Deprotection of the methyl group in 18 (1 eq.) using NaI(10 eq.) gave compound 19 after purification by flash columnchromatography.

Nucleoside 20 (7-iodo-7-deazaadenosine, 1 eq.) was reacted with linkersynthon 19 (2 eq.) in the presence of cuprous iodine (0.4 eq.),tetrakis(triphenylphosphine)palladium (0.4 eq.), and triethylamine (8eq.) in N,N-dimethylformamide for 4 hours. The reaction was concentratedand purified by flash column chromatography to afford nucleoside 21.

Phosphorous oxychloride (6 eq.) was added to nucleoside 21 (1 eq.) intrimethylphosphate at 0° C. to form the correspondingdichloromonophosphate 22. The reaction mixture was stirred at 0° C. for2 hours after which it was transferred via cannula to another flaskcontaining tributylammonium pyrophosphate (12 eq.) and tributylamine (22eq.) in the presence of DMF. After another 30 minutes at 0° C., thesolution was quenched with TEAB buffer (1M). The solution was thenstirred overnight at ambient temperature. Purification was carried outby HPLC (C-18 reverse phase) to afford nucleoside triphosphate 23.Ammonium hydroxide (28-30%) was added to dried nucleoside triphosphate23, and the resultant solution was stirred at ambient temperature for 1hour. Concentration under vacuum gave nucleotide 10 which was stored in0.1 M TEAB solution. Nucleotide 10 can be used in the synthesis ofcompound 15 as described in Example 1.

Example 3 General Coupling Reactions

For the Examples below, the following general conditions were used. Forreactions involving the coupling of an amino group to anN-hydroxysuccinimide-activated carboxyl group (NHS ester), the compoundcontaining the amino group was suspended in 100 mM TEA-bicarbonate(pH-7.0) and evaporated to dryness. The residue was then suspended in asolution of 250 mM bicarbonate (pH 9.0), and then a solution of thecompound containing the NHS ester was added. After 1 hour, the couplingreaction mixture was purified by HPLC (AX-300 anion exchange). Theproduct fractions were collected, concentrated to dryness, and purifiedby HPLC (C-18 reverse phase).

Removal of trifluoroacetate protecting groups was accomplished bytreatment with ammonium hydroxide (28-30%) at ambient temperature forone hour.

Fmoc groups were removed by heating at 55° C. for 20 minutes in thepresence of 28-30% ammonium hydroxide.

Example 4 Synthesis of Dye-Nucleotide Conjugate 33

Aminobenzoic acid 24 (1 eq.) was dissolved in fuming sulfuric acid andthe resultant solution was heated in an oil bath at 130° C. for 4 hours.The viscous solution was poured into ice and then was neutralized with aconcentrated solution of sodium bicarbonate to a pH of 8 to 9. Theaqueous solvent was removed under vacuum to afford crude compound 25which was then dissolved in aqueous solution of sodium carbonate (5%).To this solution was added FmocOSu (Example 1, 1.5 eq.) in THF. Themixture was stirred at ambient temperature for 3 hours. The resultantcrude compound 26 was purified by flash column chromatography.

N-hydroxysuccinimide and N,N-dimethylformamide were added to a slurrysolution of compound 26 (1 eq.) in methylene chloride. The solution wascooled to 0° C., followed by addition of a solution ofdicyclohexylcarbodiimide (DCC, 2 eq.) in methylene chloride. Theice-bath was removed and stirring was continued for 2 hours. The mixturewas diluted with ethyl acetate, and the organic portion was washed withaqueous solution of HCl (5%). Purified NHS ester 27 was obtained byflash column chromatography.

Nucleoside triphosphate 8 (Example 1) was reacted with dye-NHS ester 28(4′-N-trifluoroacetylaminomethyl-6-carboxyfluorescein according toprocedures generally described in Example 3 to form the expecteddye-labeled nucleotide 29. Removal of the trifluoroacetyl (TFA)protecting group (Example 3) from conjugate 29 afforded amine compound30. Reaction of compound 30 with NHS ester 27 afforded the expectedFmoc-protected compound 31.

Following removal of the Fmoc group from compound 31 to afford amine 32,rhodamine NHS ester 14 was added to afford nucleotide-dye compound 33which contains a sulfonate substituent in the linker chain between thetwo dyes.

Example 5 Synthesis of Dye-Nucleotide Conjugate 38

Nucleoside triphosphate 8 was reacted with Fmoc-protected fluoresceinNHS ester 11 to provide the expected product 34. Following removal ofthe Fmoc group to afford deprotected aminomethyl compound 35, Fmocprotected NHS ester 7 was reacted with 35 to afford Fmoc-protectedcompound 36. Removal of the Fmoc group afforded aminoethyl phosphateester compound 37 which was combined with rhodamine NHS ester 14 toafford nucleotide-dye compound 38.

Example 6 Synthesis of Dye-Nucleotide Conjugate 43

Nucleotide 10 (Example 1) was combined with TFA-protected fluoresceinNHS ester 28 to afford the expected dye-nucleotide conjugate 39.Following removal of the TFA protecting group, the resultant deprotectedproduct 40 was reacted with NHS ester 27 to afford Fmoc-protectedconjugate 41. Removal of the Fmoc group afforded amine 42, which wasreacted with rhodamine NHS ester 14 (Example 1) to afford nucleotide-dyeconjugate 43 having a phosphate diester moiety within the chain oflinker atoms between the inner dye and the nucleobase, and a sulfonatesubstituent in the linker between the two dyes.

Example 7 Alternative Route to Dye-Nucleotide Conjugate 15

NHS ester 44 (preparable by reacting dye NHS ester 14 from Example 1with p-aminomethylbenzoic acid 24 from Example 4, followed by activationof the benzoic acid with N-hydroxysuccinimide) was combined with amino40 (Example 6) to afford nucleotide-dye conjugate 15 (Example 1).

Example 8 Synthesis of Dye-Nucleotide Conjugates 51 and 57

8A. Using procedures described in Example1,5-(3-amino-1-propynyl)-2′,3′-dideoxycytidine-5′-triphosphate 45 (seeHobbs et al., supra, for synthesis) was reacted with NHS ester 7(Example 1) to afford Fmoc-protected intermediate 46. Removal of theFmoc group afforded amine 47, which was then reacted with NHS ester 11(Example 1) to afford conjugate 48. Removal of the Fmoc protecting groupafforded amine 49, which was then reacted with rhodamine NHS ester 50(e.g., see U.S. Pat. No. 5,847,162, for synthesis) to afford conjugate51.

8B. The protocol in Example 8A was carried out using5-(3-aminoethoxy-1-propynyl)-2′,3′-dideoxycytidine-5′-triphosphate 52(e.g., see U.S. Pat. No. 5,821,356 for synthesis) instead of nucleotidetriphosphate 45, to afford conjugate 57.

Example 9 Synthesis of Dye-Nucleotide Conjugate 64

Using the synthetic scheme described in Example 4, conjugate 64 wasprepared from5-(3-aminoethoxy-1-propynyl)-2′,3′-dideoxythymidine-5′-triphosphate 58(e.g., see U.S. Pat. No. 5,821,356 for synthesis) instead of nucleotide8, dye-NHS ester 28 (supra), liner NHS ester 27 (supra), and dye-NHSester 63 (e.g., prepared following methods described in U.S. Pat. No.6,080,852) instead of compound 14.

Example 10 Synthesis of Dye-Nucleotide Conjugate 69

Using procedures described in Example 5, conjugate 69 was prepared from5-(3-aminoethoxy-1-propynyl)-2′,3′-dideoxythymidine-5′-triphosphate 58(Example 9) instead of nucleotide 8, dye NHS ester 11 (supra), phosphatelinker synthon 7 (supra), and dye NHS ester 63 (Example 9) instead ofcompound 14.

Example 11 Synthesis of Dye-Nucleotide Conjugate 77

Using procedures described in Example 5, conjugate 77 was prepared from7-(3-aminoethoxy-1-propynyl)-2′,3′-dideoxy-7-deazaguanosine-5′-triphosphate70 instead of nucleotide 8, dye NHS ester 71 (prepared in the same wayas compound 11 supra, using the 5-carboxyfluorescein instead of the6-carboxyfluorescein), phosphate linker synthon 7 (supra), and dye NHSester 76 (e.g., see U.S. Pat. No. 5,847,162 for synthesis) instead ofcompound 14.

Example 12 Synthesis of Dye-Nucleotide Conjugates 82 and 85

12A. Using methods described in Example 3, sulfonate-containing linkersynthon 27 (supra) was coupled to7-(3-aminoethoxy-1-propynyl)-3′-fluoro-2′,3′-dideoxy-7-deazaguanosine-5′-triphosphate78 (e.g., see U.S. Pat. No. 5,821,356 for synthesis) to affordFmoc-protected product 79. After the Fmoc group was removed, resultantamine 80 was reacted with NHS-activated dye 81 (e.g., see U.S. Pat. No.6,051,719 for synthesis) to produce dye-labeled nucleotide 82 containinga sulfonated benzene linker.

12B. Dye-nucleotide conjugate 85 was prepared by coupling deprotectedamine 80 (Example 12A) to phosphate-containing linker synthon 7 (supra)to afford Fmoc-protected product 83. After removal of the Fmoc group,resultant amine 84 was coupled to NHS-activated dye 81 (Example 12A) toafford dye-nucleotide conjugate 85 having a linking group containingboth a sulfonate group and a phosphate group.

Example 13 Synthesis of Dye-Nucleotide Conjugate 95

Using procedures described in Example 1, methyl4-(hydroxymethyl)benzoate 86 (1.3 eq.), Amino-Link™ 1 (Example 1, 1eq.), and DMAP (0.1 eq.) were reacted together, followed by oxidationwith mCPBA (1.5 eq.) to afford phosphotriester 87. Removal of the methylgroup from the phosphotriester group using NaI (10 eq.) affordedphosphodiester 88, which was subsequently treated with LiOH (6 eq.) toremove the methyl and trifluoroacetyl protecting groups, yielding aminoacid phosphate diester 89. Protection of the amino group with FmocOSu(Example 1) gave compound 90. NHS ester 91 was obtained by treatingcompound 90 with DCC (1.3 eq.) and N-hydroxysuccinimide (3 eq.). NHSester 91 was added to nucleotide 78 (Example 12A) to form Fmoc-protectedcompound 92. After removal of the Fmoc group, resultant amine compound93 was coupled to dye 81 (Example 12A) to afford dye-nucleotideconjugate 95 containing a phosphodiester within the linker chain.

Example 14 Synthesis of Dye-Nucleotide Conjugate 106

Using procedures described in Example 2, propargyl alcohol 96(3-propyn-1-ol, 1.5 eq.), Amino-Link™ 1 (Example 1, 1 eq.), and DMAP(0.1 eq.) were reacted together, followed by oxidation with mCPBA (1.5eq.) to afford phosphotriester 97. Removal of the methyl group from thephosphotriester group using NaI (10 eq.) affordedpropargyl-trifluoroacetylaminoethyl phosphodiester 98.

Phosphodiester 98 (1.5 eq.) was reacted with 7-iodo-7-deazaadenosine 20(1 eq.) in the presence of CuI (0.4 eq.), Pd[PPh₃]₄ (0.4 eq.), andtriethylamine (8 eq.) in DMF to afford nucleoside product 99.

Phosphorous oxychloride (6 eq.) was reacted with nucleoside 99 (1 eq.)to form dichlorophosphate intermediate 100. This intermediate wastreated with tributylammonium pyrophosphate (12 eq.) and tributylamine(22 eq.), followed by hydrolysis with TEAB buffer (1 M), to formnucleotide 5′ triphosphate 101. Removal of the TFA group was achieved byammonium hydroxide to afford nucleotide amine 102.

Dye NHS ester 11 (Example 1) was added to nucleotide amine 102 in NaHCO₃buffer to afford dye-nucleotide 104. After the Fmoc group was removed,the resultant amine compound 105 was coupled to dye NHS ester 14(Example 1) to afford dye-nucleotide conjugate 106.

Example 15 Synthesis of Dye-Nucleotide Conjugate 123

Triethylsilylacetylene 108 (3 eq.) was coupled to 4-iodophenol 107 (1eq.) in the presence of CuI (0.05 eq.), Pd[PPh₃]₄ (0.05 eq.), andtriethylamine (2 eq.) in DMF. The mixture was stirred at ambienttemperature for 5 hours after which it was concentrated under vacuum toafford a crude black oil. The oil was purified by flash columnchromatography to afford pure p-(triethylsilyl)ethynyl phenol 109.

To a solution of compound 109 in dichloromethane were added triflicanhydride (trifluoromethanesulfonic anhydride, 1.2 eq.) andtriethylamine (1.2 eq.) at −40° C. After 30 minutes, the reaction wasquenched with water and the solution was extracted with dichloromethane.Purification was achieved by flash column chromatography to affordtrifluoromethanesulfonate 111.

Propargyl alcohol 110 (1.1 eq.) was coupled to compound 111 (1 eq.) inthe presence of CuI (0.1 eq.), Pd[PPh₃]₄ (0.05 eq.), and triethylamine(2 eq.) in DMF. The mixture was stirred at 60° C. for 7 hours and thenconcentrated under vacuum. Purification by flash column chromatographygave alcohol 112.

Alcohol 112 (1 eq.), Amino-Link™ 1 (1 eq.), and DMAP (0.1 eq.) werereacted together using procedures described above, followed by oxidationwith mCPBA (1.2 eq.) to produce phosphotriester 113. Removal of themethyl group from the phosphate triester group using NaI (10 eq.)afforded phosphodiester 114.

To a solution of compound 114 in tetrahydrofuran (THF) at 0° C. wasadded tetra-butylammonium fluoride (1.5 eq.). After 30 minutes, thesolution was quenched with aqueous ammonium chloride solution (10%) andextracted with ethyl acetate. Purification was achieved by flash columnchromatography to afford phosphate-containing linker synthon 115.

Compound 115 (2 eq.) was coupled to nucleoside 116(7-iodo-3′-fluoro-2′,3′-dideoxy-7-deazaguanosine, 1 eq.) (e.g., seeHobbs et al., supra, for synthesis) in the presence of CuI (0.4 eq.),Pd[PPh₃]₄ (0.4 eq.), and triethylamine (10 eq.) in DMF to affordnucleoside 117.

Phosphorous oxychloride (6 eq.) was reacted with nucleoside 117 (1 eq.)to form intermediate 118 which was treated with tributylammoniumpyrophosphate (12 eq.) and tributylamine (22 eq.), followed byhydrolysis with TEAB buffer (1 M) to form nucleotide 119. Removal of theTFA group was achieved using ammonium hydroxide to afford amine 120.

Dye 71 (supra) was added to nucleotide amine 120 in NaHCO₃ buffer toafford dye-labeled nucleotide 122 (in these examples, there is nocompound 121). After the Fmoc group was removed, the resultant aminecompound 123 was coupled with dye 76 (Example 11) to afforddye-nucleotide conjugate 124.

Example 16 Nucleotide-Dye Conjugate 137 with Phosphonate-ContainingLinker

Lithiated tert-butyl acetate 125, prepared from tert-butyl acetate andlithio N,N-diisopropylamine (prepared according to M. W. Rathke et al.,J. Amer. Chem. Soc. 95:3050 (1973)) in hexane, is added dropwise to ahexane solution of 2-cyanoethyl diisopropylchlorophosphoramidite 126(Aldrich Chemical Company) under an inert atmosphere. The reactionmixture is washed free from salts with water. The resultant organiclayer is dried over anhydrous sodium sulfate, filtered, reduced involume, and phosphoramidite 127 is isolated by column chromatography onsilica gel by elution with hexane and dichloromethane.

Phosphoramidite 127 is dissolved in dry acetonitrile and placed under aninert atmosphere. To this solution is added an acetonitrile solution of1.2 equivalents of 2-(trifluoroacetamido)ethanol 128 and 1.2 equivalentsof tetrazole. The product phosphonite 129 is isolated by columnchromatography on silica gel by elution with hexane and dichloromethane.

Phosphonite 129 is dissolved in dry dichloromethane. To the solution isadded 1.5 equivalents of tert-butyl hydroperoxide (TBHP) indichloromethane. After complete reaction, the excess TBHP is removedfrom the reaction mixture by washing with water. The resultant organiclayer is dried over anhydrous sodium sulfate, filtered, reduced involume, and the phosphonate diester product 130 is isolated by columnchromatography on silica gel by elution with hexane and dichloromethane.

Phosphonate diester 130 is dissolved in dichloromethane, and 2equivalents of tri-fluoroacetic acid (TFA) are added. After the reactionis complete, the excess TFA is removed by washing with water. Theresultant organic layer is dried over anhydrous sodium sulfate,filtered, reduced in volume, and the phosphonate diester carboxylic acid131 is isolated by column chromatography on silica gel by elution withmethanol and acetic acid in dichloromethane.

Phosphonate diester 131 is dissolved in ethyl acetate. To the solutionis added N-hydroxysuccinimide (NHS, 2 eq.) and dicyclohexylcarbodiimide(DCC, 1.2 eq.). After the reaction is complete, dicyclohexylurea isremoved by filtration, and excess NHS is removed by washing with water.The resultant organic layer is dried over anhydrous sodium sulfate,filtered, reduced in volume, and the phosphonate NHS ester 132 isisolated by column chromatography on silica gel by elution with hexaneand dichloromethane.

Phosphonate NHS ester 132 is added to nucleotide 8 (Example 1) in NaHCO₃buffer to afford nucleotide 133. After the cyanoethyl and TFA group areremoved by treatment with ammonium hydroxide, the resultant nucleotideamine 134 is coupled to dye-NHS ester 11 (Example 1) to afford theexpected dye-labeled nucleotide 135. The Fmoc group is removed, and theamine product 136 is coupled with dye-NHS ester 14 (Example 1) to afforddye-nucleotide conjugate 137.

Example 17 Synthesis of Dye-NHS Ester 14

Bicyclic amine t-Boc ester 141 (12.8 gm, 47 mmole, U.S. Pat. No.5,688,808), 1-bromo-3-chloropropane (29.3 gm, 187 mmole), sodium iodide(56.4 gm, 376 mmole) and sodium bicarbonate (7.9 gm, 94 mmole) wererefluxed together in 150 ml CH₃CN for 18 hours. The mixture was cooledto room temperature, filtered, and evaporated. The filter cake waswashed with 300 ml hexane which was combined with the filtrate andwashed with 2×50 ml water and 50 ml saturated NaCl, dried over MgSO₄,filtered, and concentrated under vacuum. The product was purified bychromatography on silica gel, eluting with hexane/ethyl acetate: 20/1,to give tricyclic amine pivalate ester 142 (pale yellow oil, 9.5 gm, 30mmole, 64% yield). The ester group of 142 was hydrolyzed in a solutionof lithium hydroxide monohydrate (2.6 gm, 60 mmole) in 15 ml water and120 ml methanol. After stirring for one hour at room temperature, themixture was concentrated under vacuum, dissolved in 30 ml 1M HCl, andextracted with 3×100 ml of diethylether. The combined ether extractswere washed with 50 ml of 200 mM pH 7 phosphate buffer, dried overMgSO₄, filtered and concentrated under vacuum to give tricyclic amine143 as a brown solid. Tricyclic amine 143 and 3,6-dichloro,4-isopropylcarboxylate phthalic anhydride 144 were refluxed in tolueneto give Friedel-Craft acylation product ketone 145 (Abs. max 400 nm).Cyclization of 145 with 143 in phosphoryl trichloride (as activatingagent) and chloroform at reflux gave dye isopropyl carboxylic ester 146as a mixture of isopropylcarboxylate regioisomers. After cleavage of theisopropyl group, dye carboxylic acid 147 was converted to NHS-rhodaminedye 14 (which can be used in Example 1).

Example 18 Synthesis of Dye-Nucleotide Conjugate 211

To a solution of 4-bromoisophthalic acid 201 (10 g, 40.8 mmol) inmethanol (150 mL) was added concentrated sulfuric acid (3.5 mL). Thesolution mixture was heated at reflux temperature for 24 hours. Afterthe reaction was complete, the methanol solvent was removed underreduced pressure. The residue was made basic with saturated sodiumbicarbonate (NaHCO₃). The solution was extracted with ethyl acetate. Theextract was washed with water and dried over sodium sulfate (Na₂SO₄).Removal of solvent gave oil, which solidified at ambient temperature.The solid was titurated twice with 20 mL of methanol and hexane (1:3) togive 8 g (90%) of 4-bromoisophthalic acid dimethyl ester 202: ¹H NMR(400 MHz, CDCl₃) δ 3.93 (s, 3H), 3.93 (s, 3H), 7.74 (d, J=8 Hz, 1H),7.95 (dd, J=2 Hz, J=8 Hz, 1H), 8.43 (d, J=2 Hz, 1H).

To a solution of 4-bromoisophthalic acid dimethyl ester 202 (2.95 g,10.8 mmol) in anhydrous DMF (50 ml), Copper(I) cyanide (1.20 g, 13.5mmol) was added in one portion. The slurry solution was heated at refluxtemperature for one hour. When the solution was cooled to ambienttemperature, it was poured into 300 mL of ammonium chloride solution(10%) at 0° C. White precipitate was formed. The slurry solution wasstirred at ambient temperature for 30 minutes. Then it was extractedwith ethyl acetate. The combined extracts were dried over sodiumsulfate. Removal of solvent gave white solid which was washed with coldmethanol to give 1.89 g (80%) of 4-cyanoisophthalic acid dimethyl ester203: ¹H NMR (400 MHz, CDCl₃) δ 3.95 (s, 3H), 4.05 (s, 3H), 7.91 (d, J=8Hz, 1H), 8.30 (dd, J=1.7 Hz, J=8 Hz, 1H), 8.77 (d, J=1.7 Hz, 1H).

4-Cyanoisophthalic acid dimethyl ester 203 (1.70 g, 7.76 mmol) wasdissolved in methanol (100 mL) and then treated with an aqueous solutionof lithium hydroxide (2.0 g, 46.5 mmol) in 50 mL of water. The basicsolution was stirred at ambient temperature for 1.5 hour. It was thenpoured into a 10% HCl solution. The solution was extracted with ethylacetate and the combined extracts were dried over Na₂SO₄. Removal ofsolvent gave quantitative yield of 4-cyanoisophthalic acid 204: ¹H NMR(400 MHz, CD₃OD) δ 8.01 (d, J=8 Hz, 1H), 8.33 (dd, J=1.7 Hz, J=8 Hz,1H), 8.73 (d, J=1.7 Hz, 1H).

A pressure flask was charged with 4-cyanoisophthalic acid 204 (1.40 g,7.33 mmol), ethanol (100 mL), Pd/C (10%) (0.50 mg), and concentrated HCl(1.5 mL). The mixture solution was hydrogenated at 50 PSI overnight. Thesolid impurities were removed by filtration. Removal of solvent gavequantitative yield of 4-aminomethylisophthalic acid 205: ¹H NMR (400MHz, CD₃OD) δ 4.46 (s, 2H), 7.69 (d, J=8 Hz, 1H), 8.26 (dd, J=2 Hz, J=8Hz, 1H), 8.76 (d, J=1.7 Hz, 1H).

A round bottom flask was charged with 4-aminomethylisophthalic acid 205,ethanol (20 mL), methanol (20 mL), excess ethyl trifluoroacetate (20mL), and triethylamine (10 mL). The mixture solution was stirred atambient temperature for 1 hour. It was then poured into a hydrochloricacid solution (10%). The solution was extracted with ethyl acetate. Thecombined extracts were dried over Na₂SO₄. Removal of solvent gavecompound 206 (1.8 g): ¹H NMR (400 MHz, CD₃OD) δ 4.9 (s, 2H), 7.51 (d,J=8 Hz, 1H), 8.17 (dd, J=2 Hz, J=8 Hz, 1H), 8.65 (d, J=2 Hz, 1H).

A 100 mL, round bottom flask was charged with crude compound 206 (1 g,3.43 mmol), anhydrous DMF (30 mL),O—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (1 g,3.43 mmol), and diisopropylethylamine (1.3 g, 10.3 mmol). The reactionsolution was stirred at ambient temperature for 30 minutes. Solvent wasremoved under reduced pressure. The crude compound was added 5% HClsolution and was extracted with ethyl acetate. The combined organicextracts were dried over Na₂SO₄. Purification was achieved by flashcolumn chromatography on silica gel with eluent CH₂Cl₂:CH₃OH (10:1)followed by CH₂Cl₂:CH₃OH (5:1) to afford 500 mg (38%) of activated ester207: H¹NMR (400 MHz, CDCl₃+10% CD₃OD) δ 2.93 (s, 4H), 4.80 (s, 2H), 7.60(d, J=8 Hz, 1H), 8.17 (dd, J=2 Hz, J=8 Hz, 1H), 8.7.0 (d, J=2 Hz, 1H);¹⁹F NMR-178 ppm (s).

Compound 208 (which is the same as compound 58 noted in Example 9) (1equiv) was dissolved in a minimum amount of formamide and a solution oflinker synthon 207 (7 equiv) in DMSO (1 mg of 207 per 5 μL of DMSO) wasadded, followed by diisopropylethylamine (20 equiv). The couplingreaction was complete in one hour at ambient temperature. Purificationwas carried out by HPLC (AX-300 anion exchange). The recovered compoundwas dried under reduced pressure and purified by HPLC (C-18 reversephase). The compound was dried under reduced pressure and then heated inammonium hydroxide (28-30%) for 15 minutes at 55° C. Then it was driedand purified by HPLC (C-18 reverse phase) to give nucleotide 209.

The following can be used as a general procedure to produce dyeconjugates from compound 209 and an activated ester, such as an NHSester of a dye (referred to as “NHS dye” in FIG. 22B). Compound 209 (1equiv) is suspended in a minimum amount of a solution of 250 mMbicarbonate (pH 9.0), and a solution of dye NHS ester 210 in DMSO (3equiv, 1 mg of dye NHS ester per 12 μL of DMSO) is then added. Thereaction mixture is placed in the dark at ambient temperature for 1hour. The product can be purified by anion exchange HPLC (AX-300) andthen C-18 reverse phase HPLC to give pure dye-labeled nucleotide 211.

All publications and patent applications mentioned herein are herebyincorporated by reference as if each publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

Although the invention has been described with reference to certainillustrative embodiments and examples, it will be appreciated thatvarious modifications and variations can be made without departing fromthe scope and spirit of the invention.

1-71. (canceled)
 72. A nucleic acid sequencing kit comprising at leastone labeled nucleoside triphosphate comprising a conjugate comprising adye labeled nucleobase of the form: (1) B-L-D, wherein B is anucleobase, L is an anionic linker, and D comprises at least onefluorescent dye that comprises a xanthene, a rhodamine or a fluorescein,or (2) B-L1-D1-L2-D2, wherein B is a nucleobase, L1 and L2 are linkerssuch that at least one of L1 and L2 is an anionic linker, and D1 and D2are members of a fluorescent donor/acceptor pair, such that one of D1and D2 is a donor dye capable of absorbing light at a first wavelengthand emitting energy in response thereto, and the other of D1 and D2 isan acceptor dye capable of absorbing energy emitted by the donor dye andfluorescing at a second wavelength in response thereto, and at least oneof D1 and D2 comprises a xanthene, a rhodamine or a fluorescein, whereinL or at least one of L1 and L2 comprises at least one anionic phosphateor anionic phosphate and one or more of the following components: a3′-extendable primer, a polymerase enzyme, one or more 3′ extendablenucleotides which are not labeled with conjugate, and/or a bufferingagent.
 73. The kit of claim 72 wherein at least one labeled nucleosidetriphosphate is nonextendable.
 74. The kit of claim 72 which comprisesfour different labeled nucleoside triphosphates which are complementaryto A, C, T and C, and each of which contains a distinct conjugate. 75.The kit of claim 74 wherein the four different labeled nucleosidetriphosphates are nonextendable.
 76. The kit of claim 74 wherein thefour different labeled nucleoside triphosphates are extendableribonucleoside triphosphates.
 77. The kit of claim 72 wherein theconjugate has the form B-L1-D1-L2-D2.
 78. The kit of claim 77 whereinthe donor dyes in the four different labeled nucleoside triphosphatesare the same.
 79. The kit of claim 78 wherein the donor dye is anorthocarboxyfluorescein.
 80. The kit of claim 78 wherein the donor dyeis a 4,7-dichloro-orthocarboxyfluorescein. 81-85. (canceled)
 86. The kitof claim 77 wherein L2 is an anionic linker and L1 is not an anioniclinker.
 87. The kit of claim 77 wherein L2 comprises a carboxylic acidmoiety.
 88. The kit of claim 87 wherein the carboxylic acid moiety is acarboxy benzene moiety.
 89. The kit of claim 72 wherein the dye-labelednucleobase is of the form B-L-D.
 90. The kit of claim 89 wherein Lcomprises a sulfonic acid moiety.
 91. The kit of claim 89 wherein Lcomprises a sulfonated benzene moiety.
 92. The kit of claim 89 wherein Lcomprises an anionic phosphate moiety.
 93. The kit of claim 92 whereinthe anionic phosphate moiety is a phosphate diester moiety, and thephosphorus atom of the phosphate diester moiety is located in L within achain of linker atoms that connect B to D.
 94. The kit of claim 89wherein L comprises an anionic phosphonate moiety.
 95. The kit of claim94 wherein the anionic phosphonate moiety is a phosphonate monoestermoiety, and the phosphorus atom of the phosphonate monoester moiety islocated in L within a chain of linker atoms that connect B to D.
 96. Thekit of claim 89 wherein L comprises a carboxylic acid moiety.
 97. Thekit of claim 96 wherein the carboxylic acid moiety is a carboxyl benzenemoiety.
 98. The kit of claim 89 wherein L comprises 4 to 20 chain atoms.99. The kit of claim 89 wherein D comprises at least one fluorescein orrhodamine.
 100. The kit of claim 72 wherein B comprises adenine,7-deazaadenine, 7-deaza-8-azaadenine, cytosine, guanine, 7-deazaguanine,7-deaza-8-azaguanine, thymine, uracil, or inosine.